Compositions and methods for diminishing an immune response

ABSTRACT

The invention is based upon the discovery that T regulatory type 1 (Tr1) cells express particular cell surface markers that allow for their selection, enrichment, isolation, purification and administration. The ability to use the particular markers described herein to select, enrich, isolate, purify and administer Tr1 cells allows for improved methods of Tr1 therapies for treating a wide variety of diseases and disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.15/711,194, filed Sep. 21, 2017 which is a Divisional of U.S. patentapplication Ser. No. 14/407,627, filed Dec. 12, 2014, which in turn is a371 of PCT International Application No. PCT/US2013/046378, filed Jun.18, 2013, which claims the benefit of U.S. Provisional Application Ser.No. 61/661,172, filed Jun. 18, 2012, and U.S. Provisional ApplicationSer. No. 61/816,497 filed Apr. 26, 2013, the contents of each of whichare incorporated herein by reference.

BACKGROUND OF THE INVENTION

T regulatory type 1 (Tr1) cells were discovered in peripheral blood ofsevere combined immunodeficiency patients with long-term mixed chimerismafter HLA-mismatched fetal liver hematopoietic stem cell transplant(HSCT) (Roncarolo et al., 1988, J Exp Med 167, 1523-1534; Bacchetta etal., 1994, J Exp Med 179, 493-502). Tr1 cells have strongimmunosuppressive capacity in several immune-mediated diseases(Roncarolo and Battaglia, 2007, Nat Rev Immunol 7, 585-598; Roncarolo etal., 2011, Immunol Rev 241, 145-163; Pot et al., 2011, Semin Immunol 23,202-208). The secretion of high levels of IL-10, and the killing ofmyeloid antigen-presenting cells (APCs) via Granzyme B are the mainmechanisms of Tr1-mediated suppression (Groux et al., 1997, Nature 389,737-742; Magnani et al., 2011 Eur J Immunol 41, 1652-1662). To datespecific biomarkers for Tr1 cells have not been identified, limitingtheir study and clinical application. Tr1 cells are distinguished from Thelper (T_(H))1, T_(H)2, and T_(H)17 cells by their unique cytokineprofile and the regulatory function. Tr1 cells secrete higher levels ofIL-10 than IL-4 and IL-17, the hallmark cytokines of T_(H)2 and T_(H)17cells, respectively. Tr1 cells also secrete low levels of IL-2 and,depending on the local cytokine milieu, can produce variable levels ofIFN-γ, together, the key T_(H)1 cytokines (Roncarolo et al., 2011,Immunol Rev 241, 145-163). FOXP3 is not a biomarker for Tr1 cells sinceits expression is low and transient upon activation. IL-10-producing Tr1cells express ICOS (Haringer et al., 2009, J Exp Med 206, 1009-1017) andPD-1 (Akdis et al., 2004, J Exp Med 199, 1567-1575), but these markersare not specific (Maynard et al., 2007, Nat Immunol 8, 931-941). CD49b,the α2 integrin subunit of the very-late-activation antigen (VLA)-2, hasbeen proposed as a marker for IL-10-producing T cells (Charbonnier etal., 2006, J Immunol 177, 3806-3813); but it is also expressed by humanT_(H)17 cells (Boisvert et al., 2010, Eur J Immunol 40, 2710-2719).Moreover, murine CD49b⁺ T cells secrete IL-10 (Charbonnier et al., 2006,J Immunol 177, 3806-3813) but also pro-inflammatory cytokines (Kassiotiset al., 2006, J Immunol 177, 968-975). Lymphocyte activation gene-3(LAG-3), a CD4 homolog that binds with high affinity to MHC class IImolecules, is expressed by murine IL-10-producing CD4⁺ T cells (Okamuraet al., 2009, Proc Natl Acad Sci USA 106, 13974-13979), but also byactivated effector T cells (Workman and Vignali, 2005, J Immunol 174,688-695; Bettini et al., 2011, J Immunol 187, 3493-3498; Bruniquel etal., 1998, Immunogenetics 48, 116-124; Lee et al., 2012, Nat Immunol 13,991-999) and by FOXP3⁺ regulatory T cells (Tregs) (Camisaschi et al.,2010, J Immunol 184, 6545-6551). It was recently shown that human Tr1cells express CD226 (DNAM-1), which is involved in the specific killingof myeloid APCs (Magnani et al., 2011 Eur J Immunol 41, 1652-1662).Overall, none of the abovementioned markers has been confirmed to beselective for Tr1 cells.

Tr1 cell-based clinical approaches are still largely limited by theinability to transfer a pure population of these cells. Moreover, a highfrequency of Tr1 cells has been correlated with a positive outcome afterHSCT (Bacchetta et al., 1994, J Exp Med 179, 493-502; Serafini et al.,2009, Haematologica 94, 1415-1426), but the absence of suitable markershas made the clinical screening of this type of Tr1 cells impossible.Hence, the availability of specific biomarkers of Tr1 cells wouldfacilitate the transition of therapies targeting Tr1 cells from bench tobedside.

Thus, there is a need in the art for compositions and methods toidentify and purify Tr1 cells. The present invention satisfies thisunmet need.

SUMMARY OF THE INVENTION

The invention described herein is based in part upon the discovery thatT regulatory type 1 (Tr1) cells express particular cell surface markersthat allow for their selection, enrichment, isolation, purification andadministration. In one embodiment, the invention is a compositioncomprising an enriched population of T regulatory type 1 (Tr1) cells,wherein the Tr1 cells in the enriched population of Tr1 cells expressthe cell surface markers CD4, and CD49b, and LAG-3. In some embodiments,the Tr1 cells also express the cell surface marker CD226. In someembodiments, the Tr1 cells express the cell surface marker CD226 at alevel greater than the level of CD226 expressed by a comparator cellpopulation. In various embodiments, the comparator cell population is atleast one selected from the group consisting of CD49b-LAG-3− T cells andTH0 cells. In some embodiments, the Tr1 cells do not constitutivelyexpress high levels of Foxp3, as compared with the level of Foxp3 on acomparator cell selected from the group consisting of a CD25bright Tcell and a Foxp3+ Treg cell. In one embodiment, greater than 90% of thecells in the enriched population of Tr1 cells express the cell surfacemarkers CD4, and CD49b, and LAG-3. In another embodiment, greater than95% of the cells in the enriched population of Tr1 cells express thecell surface markers CD4, and CD49b, and LAG-3. In another embodiment,greater than 98% of the cells in the enriched population of Tr1 cellsexpress the cell surface markers CD4, and CD49b, and LAG-3. In anotherembodiment, wherein greater than 99% of the cells in the enrichedpopulation of Tr1 cells express the cell surface markers CD4, and CD49b,and LAG-3.

In another embodiment, the invention is a method of isolating anenriched population of Tr1 cells from a biological sample of a subjectincluding the steps of obtaining a T cell-containing biological sampleof a subject, and isolating cells from the biological sample of thesubject that express the cell surface markers CD4, CD49b, and LAG-3. Insome embodiments, the method includes the additional step of removingcells that express high levels of Foxp3 from the enriched population ofTr1 cells. In some embodiments, the method includes the additional stepof isolating cells from the biological sample of the subject thatexpress the cell surface marker CD226. In some embodiments, the cellsexpress the cell surface marker CD226 at a level greater than the levelof CD226 expressed by a comparator cell population. In one embodiment,the comparator cell population is at least one selected from the groupconsisting of CD49b-LAG-3− T cells and TH0 cells. In some embodiments,greater than 90% of the cells in the enriched population of Tr1 cellsexpress the cell surface markers CD4, and CD49b, and LAG-3. In someembodiments, greater than 95% of the cells in the enriched population ofTr1 cells express the cell surface markers CD4, and CD49b, and LAG-3. Insome embodiments, greater than 98% of the cells in the enrichedpopulation of Tr1 cells express the cell surface markers CD4, and CD49b,and LAG-3. In some embodiments, greater than 99% of the cells in theenriched population of Tr1 cells express the cell surface markers CD4,and CD49b, and LAG-3. In one embodiment, the step of isolating cellsfrom the biological sample of the subject employs the use of antibodythat specifically binds to a cell surface marker. In variousembodiments, the cell surface marker is at least one selected from thegroup consisting of CD4, CD49b, and LAG-3. In some embodiments, the stepof cells from the biological sample of the subject employs the use offluorescence-activated cell sorting (FACS). In various embodiments, thebiological sample is at least one selected from the group consisting ofblood, bone marrow, cord blood, lymph, thymus, and spleen.

In one embodiment, the invention is a method of treating or preventing adisease or disorder in a subject in need thereof, the method comprisingadministering to the subject an effective amount of Tr1 cells thatexpress the cell surface markers CD4, and CD49b, and LAG-3. In someembodiments, the disease or disorder is at least one selected from thegroup consisting of an inflammatory disease and disorder, an autoimmunedisease or disorder, and a disease or disorder associated withtransplantation. In other embodiments, the disease or disorder is atleast one selected from the group consisting of allergy, asthma,inflammatory bowel disease, autoimmune entheropathy, Addision's disease,alopecia areata, ankylosing spondylitis, autoimmune hepatitis,autoimmune parotitis, Crohn's disease, diabetes mellitus, dystrophicepidermolysis bullosa, epididymitis, glomerulonephritis, Graves'disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia,systemic lupus erythematosus, multiple sclerosis, myasthenia gravis,pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis,sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies,thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia,ulcerative colitis, cell and organ transplant rejection and graft versushost disease. In one embodiment, the subject is human.

In another embodiment, the invention is a method of inhibitingalloreactive T cells in a subject in need thereof, the method includingthe step of contacting the alloreactive T cells with an effective amountof Tr1 cells that express the cell surface markers CD4, and CD49b, andLAG-3.

In another embodiment, the invention is a method of inhibiting a T cellmediated immune response in a subject in need thereof, the methodincluding the step of contacting at least one T-lymphocyte with aneffective amount of Tr1 cells that express the cell surface markers CD4,and CD49b, and LAG-3. In some embodiments, the inhibited T cell mediatedimmune response is an effector T cell activity and the at least oneT-lymphocyte is a CD4+T-lymphocyte. In some embodiments, the inhibited Tcell mediated immune response is a cytotoxic T-lymphocyte (CTL) activityand the at least one T-lymphocyte is a cytotoxic T-lymphocyte.

In one embodiment, the invention is a method of generating animmunomodulatory effect in a subject having an alloreactive response,inflammatory response, or autoimmune response, including the step ofadministering to said subject an effective amount of CD4+CD49+LAG-3+ Tr1cells.

In another embodiment, the invention is a method of preventing ortreating an alloreactive response, inflammatory response, or autoimmuneresponse in a subject, including the step of administering to saidsubject, prior to onset of the alloreactive response, inflammatoryresponse, or autoimmune response, an effective amount of CD4+CD49+LAG-3+Tr1 cells to prevent said response.

In one embodiment, the invention is a composition comprisingCD4+CD49+LAG-3+Tr1 cells for use in treating or preventing a disease ordisorder in a subject in need thereof, wherein the disease or disorderis at least one selected from the group consisting of an inflammatorydisease and disorder, an autoimmune disease or disorder, and a diseaseor disorder associated with transplantation.

In another embodiment, the invention is a composition comprisingCD4+CD49+LAG-3+ Tr1 cells for use in treating or preventing a disease ordisorder in a subject in need thereof, wherein the disease or disorderis at least one selected from the group consisting of allergy, asthma,inflammatory bowel disease, autoimmune entheropathy, Addision's disease,alopecia areata, ankylosing spondylitis, autoimmune hepatitis,autoimmune parotitis, Crohn's disease, diabetes mellitus, dystrophicepidermolysis bullosa, epididymitis, glomerulonephritis, Graves'disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia,systemic lupus erythematosus, multiple sclerosis, myasthenia gravis,pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis,sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies,thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia,ulcerative colitis, cell and organ transplant rejection and graft versushost disease.

In one embodiment, the invention is a composition comprisingCD4+CD49+LAG-3+Tr1 cells for use in inhibiting alloreactive T cells in asubject in need thereof.

In another embodiment, the invention is a composition comprisingCD4+CD49+LAG-3+ Tr1 cells for use in inhibiting a T cell mediated immuneresponse in a subject in need thereof. In some embodiments, theinhibited T cell mediated immune response is an effector T cellactivity. In other embodiments, the inhibited T cell mediated immuneresponse is a cytotoxic T-lymphocyte (CTL) activity.

In one embodiment, the invention is a composition comprisingCD4+CD49+LAG-3+Tr1 cells for use in generating an immunomodulatoryeffect in a subject having an alloreactive response, inflammatoryresponse, or autoimmune response, the method comprising administering tosaid subject an effective amount of CD4+CD49+LAG-3+ Tr1 cells.

In another embodiment, the invention is a composition comprisingCD4+CD49+LAG-3+ Tr1 cells for use in preventing or treating analloreactive response, inflammatory response, or autoimmune response ina subject, said method comprising administering to said subject, priorto onset of the alloreactive response, inflammatory response, orautoimmune response, an effective amount of CD4+CD49+LAG-3+ Tr1 cells toprevent said response.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1, comprising FIG. 1A through FIG. 1D, depicts the results ofexperiments demonstrating the identification of CD49b, LAG-3 and CD226by gene expression profile of human Tr1 cell clones. Tr1 and TH0 cellclones were isolated from peripheral blood of 2 Healthy Donors (HDs).mRNA from T cell clones unstimulated (t0, n=4 Tr1 cell clones and n=10T_(H)0 cell clones) or stimulated with immobilized anti-CD3 and solubleanti-CD28 mAbs (6 h and 16h, n=4 Tr1 cell clones and n=5 T_(H)0 cellclones) was isolated. Differential expression of 28869 genes wasinvestigated by whole transcript Affymetric chips. FIG. 1A: Followingdata normalization by standard Robust Multichip Analysis (RMA) protocoland statistical analysis (t test welch without the False Discovery Rate,FDR, correction), Tr1 and T_(H)0 cell populations were compared at thethree time points. Normalized expression values for profiles directlycomparing Tr1 vs. T_(H)0 cell clones at t0, 6 h and 16 h are shown. FIG.1B-1C: Two-dimensional heatmaps of genes differentially expressed (DEGs)encoding for membrane proteins in Tr1 as compared to T_(H)0 cell clones.Heatmap of DEGs in Tr1, as compared to T_(H)0 cell clones, at the threetime points (t0, t6h, and t16h) (FIG. 1B) and at 6h and 16 h (FIG. 1C)are shown. Red genes are expressed at higher levels compared to the meansignal intensities in all experiments, whereas down-regulated genes arein green, and in black are signal intensities close to the meanexpression level. The rows are scaled to have mean zero and standarddeviation one. Gene Name, and Gene Symbol are indicated. (FIG. 1D)Expression of CD49b, LAG-3, and CD226 measured by flow cytometry in Tr1and T_(H)0 cell clones. Percentages of CD49b⁺, and of LAG-3⁺, and meanfluorescence intensity (MFI) of CD226 in Tr1 and T_(H)0 cell clones arepresented. P=**p≤0.005, ****p<0.0001.

FIG. 2, comprising FIG. 2A through FIG. 2C, depicts the results ofexperiments demonstrating that the co-expression of CD49b and LAG-3identifies human Tr1 cells in vivo in healthy donors. FIG. 2A:Expression of CD49b and LAG-3 (gated on CD4⁺CD45RA⁻ T cells) in blood ofHDs. Dot plots of 1 representative donor out of 23 donors are presented(left and middle panels); percentages of cells in each quadrant areindicated. Percentages of CD49b⁺LAG-3⁻, CD49b⁻LAG-3⁺, and CD49b⁺LAG-3⁺ Tcells in each donor analysed are shown (right panel). FIG. 2B:Concentration levels of IL-10, IL-4, IFN-γ and IL-17 in culturesupernatants of the indicated FACS-sorted T cell populations stimulatedwith antibodies to CD3 and CD28. Mean±SEM; n=9 (IL-10, IL-4 and IFN-γ)and n=4 (IL-17). Ratios of IL-10 vs. IL-4, IFN-γ and IL-17 in 1representative donor out of 9 tested for IL-10/IL-4 and IL-10/IFN-γ, and4 tested for IL-10/IL-17 are shown. *P≤0.05, **P≤0.005. When notindicated differences were not statistically different. FIG. 2C:Suppression mediated by the indicated FACS-sorted T cell populations.One representative experiment out of 6 (left panel), and percentages ofsuppression in 6 independent experiments are shown (right panel).P=*p≤0.05, ** P≤0.005.

FIG. 3, comprising FIG. 3A through FIG. 3D, depicts the results ofexperiments demonstrating that co-expression of CD49b and LAG-3identifies Tr1 cells in anti-CD3 treated mice. T cells were isolatedfrom the small intestine of anti-CD3 treated mice. FIG. 3A: Expressionof CD49b and LAG-3 measured on CD4⁺ TCRβ⁺Foxp3^(RFP−)IL-10^(eGFPbright)or IL-10^(eGFP−) T cells (left panel). Percentages of cells in eachquadrant are indicated. Frequencies (mean±SEM) of cells co-expressingCD49b and LAG-3 among Tr1 cells (defined as CD4⁺TCRβ⁺Foxp3^(RFP−)IL-10^(eGFPbright)) and CD4⁺IL-10⁻ T cells (defined asCD4⁺ TCRβ⁺IL-10^(eGFP−)) obtained in 5 independent experiments (rightpanel) are shown. P=**p≤0.005. FIG. 3B: IL-10^(eGFP) frequency and MFIin the indicated T cell populations (gated on CD4⁺ TCRβ⁺Foxp3^(RFP−))isolated from the small intestine of anti-CD3 treated mice.Representative dot plots from 1 experiment out of 5 are shown (leftpanel). In each experiment 2 to 5 mice were pooled. Percentages of cellsin each quadrant are indicated. MFI for IL-10^(eGFP+) T cells in theindicated T cell populations are shown (right panel). FIG. 3C: Mean±SEMof IL-10^(eGFP+)cell frequencies among the indicated T cell populationsobtained in 5 independent experiments is shown. ** P≤0.005. FIG. 3D:Concentration levels of IL-10, IL-4, IFN-γ and IL-17A in culturesupernatants of the indicated FACS-sorted T cell populations (gated onCD4⁺ TCRβ⁺Foxp3^(RFP−)) from the small intestine of anti-CD3 treatedmice (Mean±SEM) and the ratios of IL-10 vs. IL-4, IFN-γ and IL-17A in 1representative experiment out of 3 are shown. In each experiment cellsisolated from 5 mice were pooled before FACS-sorting. Each experimentcontains at least 3 replicates of the same sample for each population. *P≤0.05, ** P≤0.005, *** P≤0.0005. When not indicated differences werenot statistically different.

FIG. 4, comprising FIG. 4A through FIG. 4E, depicts the results ofexperiments demonstrating the in vitro and in vivo regulatory activityof murine CD4⁺CD49b⁺LAG-3⁺ T cells. FIG. 4A: Suppression mediated by theindicated FACS-sorted T cell populations isolated from the smallintestine of anti-CD3 treated mice. One representative experiment out of3 (left panel), and mean±SEM of the percentages of suppression in 3independent experiments (right panel) are shown. P=**p≤0.005. When notindicated differences were not statistically different. FIG. 4B:eT_(H)17 (CD4⁺ TCRβ⁺Foxp3^(RFP−)IL-17A^(eGFP+)) cells were isolated fromthe colon and mesenteric lymph nodes of RAG1^(−/−) mice injected withCD4⁺CD45RB^(High) T cells isolated from Foxp3^(RFP)IL-17A^(eGFP) doublereporter mice. The indicated T cell populations were isolated from thesmall intestine of anti-CD3-treated mice and injected i.p. incombination with eT_(H)17 (ratio 1:1) into RAG1^(−/−) mice. FIG. 4C:Representative endoscopic (upper panels) and histological (lower panels)pictures. Endoscopic (FIG. 4D), mass loss (FIG. 4E) colitis score weremeasured. Each dot represents 1 mouse. Lines indicate mean±SEM. **P≤0.005, and *** P≤0.0005.

FIG. 5, comprising FIG. 5A through FIG. 5F, depicts the results ofexperiments demonstrating that co-expression of CD49b and LAG-3 isspecific for murine Tr1 cells. FIG. 5A: Expression of CD49b and LAG-3measured on CD4⁺ TCRβ⁺IL-4^(eGFP+) (T_(H)2), CD4⁺ TCRβ⁺Foxp3^(RFP−)IL-17A^(eGFP+)(T_(H)17), and CD4⁺ TCRβ⁺Foxp3^(RFP+)IL-17A^(eGFP−)(Foxp3⁺ Tregs) cells isolated from the draining lymph nodes ofIL-4^(eGFP) and Foxp3^(RFP)IL-17A^(eGFP) mice 10 days after N.brasiliensis infection. Dot plots from 1 representative experiment outof 3 are shown. Percentages of cells in each quadrant are indicated.FIG. 5B: Expression of CD49b and LAG-3 on CD4⁺TCRβ⁺Foxp3^(RFP−)IL-10^(eGFPbright) T cells isolated from the draininglymph nodes of Foxp3^(RFP)IL-10^(eGFP) mice infected with N.brasiliensis. Dot plots from 1 representative experiment out of 4 areshown. Percentages of cells in each quadrant are indicated. FIG. 5C:Mean±SEM of frequencies of cells co-expressing CD49b and LAG-3 among theindicated T cells isolated from the draining lymph nodes of infectedmice (n=5 for each groups). ***P≤0.0005. FIG. 5D: Frequencies ofCD4⁺IL-10^(eGFP+) T cells and IL-10^(eGFP) MFI in the indicated T cellpopulations (gated on CD4⁺ TCRβ⁺Foxp3^(RFP−)) from the draining lymphnodes of Foxp3^(RFP)IL-10^(eGFP) mice 10 days after N. brasiliensisinfection. Dot plots from 1 representative experiment out of 4 areshown. Percentages of cells in each quadrant are indicated. FIG. 5E:Mean±SEM of IL-10^(eGFP+)cell frequencies among the indicated T cellpopulations isolated from the draining lymph nodes of infectedFoxp3^(RFP)IL-10^(eGFP) mice (n=5 for each groups). *P≤0.05,***P≤0.0005. FIG. 5F: Suppression mediated by the indicated FACS-sortedT cell populations from the draining lymph nodes of infectedFoxp3^(RFP)IL-10^(eGFP) mice. One representative experiment out of 3(left panel) and mean±SEM of the percentages of suppression obtained in3 independent experiments (the right panel) are shown. *P≤=0.05 and**P≤0.005.

FIG. 6, comprising FIG. 6A through FIG. 6F, depicts the results ofexperiments demonstrating that co-expression of CD49b and LAG-3 allowsthe selection of human Tr1 cells in vitro and the enumeration of Tr1cells in vivo in tolerant subjects. FIG. 6A: Percentages ofCD49b⁺LAG-3⁻, CD49b⁻LAG-3⁺, and CD49b⁺LAG-3⁺cells in Tr1 (pTr1) andT_(H)0 cell lines polarized with artificial APC. **P≤0.005. When notindicated differences were not statistically different. FIG. 6B:Percentages of CD49b⁺LAG-3⁻, CD49b⁻LAG-3⁺, and CD49b⁺LAG-3⁺cells inpTr1(DC-10) and T(mDC) cell lines polarized with DC. **P≤0.005 and***P≤0.0005. When not indicated differences were not statisticallydifferent. FIG. 6C: IL-10 levels in culture supernatants of pTr1 celllines and of FACS-sorted CD49b⁺LAG-3⁺ T cells from pTr1 cells(CD49b⁺LAG-3⁺pTr1). Mean±SEM (n=4). *P≤0.05. FIG. 6D: Suppressionmediated by pTr1 cells and CD49b⁺LAG-3⁺ T cells sorted from pTr1 cells(CD49b⁺LAG-3⁺pTr1). One representative experiment out of 5 (left panel),and percentages of suppression in 5 independent experiments (rightpanel) are shown. **P≤0.005. FIG. 6E: Expression of CD49b and LAG-3(gated on CD4⁺CD45RA⁻ T cells) in subjects with complete chimerism (CC)and persistent mixed chimerism (PMC) after allogeneic HSCT.Representative dot plots from 1 out of 7 CC and 1 out 11 PMC are shown,percentages of cells in each quadrant are indicated. FIG. 6F:Percentages of CD49b⁺LAG-3⁻, CD49b⁻ LAG-3⁺, and CD49b⁺LAG-3⁺ T cells ineach healthy donor (HD) and transplanted subjects analysed. *P≤0.05,**P≤0.005, *** P≤0.0005. When not indicated differences were notstatistically different.

FIG. 7, comprising FIG. 7A through FIG. 7D, depicts the results ofexperiments demonstrating the validation and selection of genes encodingfor CD49b, CD226, and LAG-3. Tr1 and T_(H)0 cell clones, isolated fromperipheral blood of 2 Healthy Donors (HDs). mRNA from cells unstimulated(t0, n=4 Tr1 cell clones and n=10 T_(H)0 cell clones) or stimulated withimmobilized anti-CD3 and soluble anti-CD28 mAbs (6 h and 16h, n=4 Tr1cell clones and n=5 T_(H)0 cell clones) was isolated FIG. 7A: Expressionof IL-10, GZB, and PD1 determined by the DNA microarray is shown. *P0.05 and **P 0.005. FIG. 7B: Expression of CD49b, CD226, and LAG-3determined by the DNA microarray is shown. **P 0.005. When not indicateddifferences were not statistically different. FIG. 7C: Expression ofCD49b, CD226, and LAG3 in T_(H)0 and Tr1 cell clones. Followingnormalization to HPRT and B2M, relative mRNA amounts of T cell cloneswere adjusted to corresponding expression levels of a calibrator (poolof CD4⁺ T cell lines from 4 HDs). Numbers represent arbitrary units. **P0.005 and ***P 0.0005. When not indicated differences were notstatistically different. FIG. 7D: IL-10-producing cells purified frompTr1 and T_(H)0 cell lines were stimulated for 6 h with immobilizedanti-CD3 and soluble anti-CD28 mAbs. Expression of the indicated geneswas investigated by RT-PCR. Following normalization to HPRT, relativemRNA amounts of T cells were adjusted to corresponding expression levelsof a calibrator (pool of CD4⁺ T cell lines from 4 HDs). Numbersrepresent arbitrary units. *P 0.05.

FIG. 8, comprising FIG. 8A through FIG. 8C, depicts the results ofexperiments demonstrating that CD49b⁺LAG-3⁺ T cells are CD25^(low) anddo not express FOXP3. FIG. 8A: Expression of CD226 in the indicated Tcell populations in peripheral blood of Healthy Donors (HDs). Meanfluorescent intensity (MFI) of CD226 expressed in the indicated T cellpopulations (gated on CD4⁺CD45RA⁻ T cells) (left panel) and mean±SEM ofthe CD226 MFI in the indicated T cell populations relative to the CD226MFI of CD49b⁻LAG-3⁻ T cells obtained in 7 donors (right panel) arereported. **P 0.005. FIG. 8B: Expression of CD25 in CD4⁺CD45RA⁻FOXP3⁺ Tcells and CD4⁺CD45RA⁻CD49b⁺LAG-3⁺ T cells and of FOXP3 inCD4⁺CD45RA⁻CD25^(bright) and CD4⁺CD45RA⁻CD49b⁺LAG-3⁺ T cells. Onerepresentative donor out of 4 is shown; numbers in histograms indicateMFI (gated on CD4⁺CD45RA⁻FOXP3⁺or CD4⁺CD25^(bright) T cells in blue, andon CD4⁺CD45RA⁻CD49b⁺LAG-3⁺ T cells in red). Expression of FOXP3(normalized to HPRT) measured by RT-PCR in the indicated FACS-sorted Tcell populations from peripheral blood of HDs. One representative donorout of 3 to 5 and mean±SEM of 3-5 independent donors is shown. **P0.005. When not indicated differences were not statistically different.FIG. 8C: IL-10/IL-4 ratio in the indicated FACS-sorted T cellpopulations activated with immobilized anti-CD3 and soluble anti-CD28mAbs for 72 h are shown. Three out of 9 donors tested.

FIG. 9, comprising FIG. 9A through FIG. 9D, depicts the results ofexperiments demonstrating that co-expression of CD49b and LAG-3identifies murine Tr1 cells in anti-CD3 treated mice. FIG. 9A:Expression of CD226. MFI of CD226 expressed by the indicated T cellpopulations (gated on CD4⁺ TCRβ⁺Foxp3^(RFP−) T cells) analyzed 4 h afterthe second anti-CD3 mAb injection (upper panel) and MFI of CD226expressed in the indicated T cell populations (gated on CD4⁺TCRβ⁺Foxp3^(RFP−) T cells) relative to the expression ofCD4⁺CD49b⁻LAG-3⁻ T cells (lower panel) is shown. *P 0.05. When notindicated differences were not statistically different. FIG. 9B:Frequency of the indicated T cell populations (gated on CD4⁺TCRβ⁺Foxp3^(RFP−) T cells) at 4 h (52), 48 h (100) and 96 h (144) afterthe second anti-CD3 mAb injection. FIG. 9C: Expression of the Il10, Il4,Ifng, Il17a, Il2, Tnfa (normalized to Hprt) measured by RT-PCR in theindicated FACS-sorted T cell populations from the small intestine ofanti-CD3 treated mice. As controls, T_(H)1 (CD4⁺ TCRβ⁺IFN-γ^(Katushka+))T_(H)17 (CD4⁺ TCRβ⁺IL-17A^(eGFP+)) and Foxp3⁺ Treg (CD4⁺TCRβ⁺Foxp3^(RFP+)) cells isolated from the small intestine ofFoxp3^(RFP)IFN-γ^(Katushka) and Foxp3^(RFP)IL-17A^(eGFP) reporter miceinjected with anti-CD3 mAb were used. Mean±SEM of 3 independentexperiments. *P 0.05, **P 0.005, and ***P 0.0005 vs. CD4+CD49b+LAG-3+ Tcells. When not indicated differences were not statistically different.FIG. 9D: The indicated FACS-sorted T cell populations from the smallintestine of anti-CD3 treated mice re-stimulated in vitro with anti-CD3and anti-CD28 mAbs for 72 h were tested for cytokine production.Mean±SEM of IL-2 and TNF-α and the ratios of IL-10 vs. IL-2 and TNF-αare presented. One representative experiment out of 3. In eachexperiment cells isolated from 5 mice were pooled before FACS-sorting.*p 0.05. When not indicated differences were not statisticallydifferent.

FIG. 10 depicts the results of experiments demonstrating thatCD4⁺CD49b⁺LAG-3⁺ T cells express AhR. Expression of the indicatedtranscription factors (normalized to Hprt) measured by RT-PCR in theindicated FACS-sorted T cell populations from the small intestine ofanti-CD3 treated mice. As controls, T_(H)1 (CD4⁺TCRβ⁺IFN-γ^(Katushka+)), T_(H)17 (CD4⁺ TCRβ⁺IL-17A^(eGFP+)) and Foxp3⁺Tregs (CD4⁺ TCRβ⁺Foxp3^(RFP+)) isolated from the small intestine ofFoxp3^(RFP)IFN-γ^(Katushka) and Foxp3^(RFP)IL-17A^(eGFP) reporter miceinjected with anti-CD3 mAb were used. Mean±SEM of 3 independentexperiments is shown. *P 0.05, **P 0.005, and ***P 0.0005 vs.CD4⁺CD49b⁺LAG-3⁺ T cells. When not indicated differences were notstatistically different.

FIG. 11 depicts the results of experiments demonstrating thatCD4⁺CD49b⁺LAG-3⁺ T cells suppress T cell responses in vitro in adose-dependent manner. Foxp3^(RFP)IL-10^(eGFP) double reporter mice wereinjected i.p. with anti-CD3 mAb at 0 and 48 h. CD4⁺TCRβ⁺Foxp3^(RFP−)CD49b⁺LAG-3⁺ T cells were FACS-sorted from the smallintestine of anti-CD3 treated mice 4 h after the second injection andtested for their ability to suppress the proliferation of responder CD4⁺T cells in vitro at the indicated cells ratios. Percentages ofsuppression are indicated. One representative experiment out of 2 isshown.

FIG. 12, comprising FIG. 12A through FIG. 12C, depicts the results ofexperiments demonstrating that the in vivo regulatory activity of murineCD4⁺CD49b⁺LAG-3⁺ T cells is IL-10 dependent. FIG. 12A: eT_(H)117 (CD4⁺TCRβ⁺Foxp3^(RFP−)IL-17A^(eGFP+)) and Dominant Negative IL-10R−eT_(H)17(DNR eTH17) cells were isolated from the colon and mesenteric lymphnodes of RAG1^(−/−) mice injected with CD4⁺CD45RB^(High) T cellsisolated from either Foxp3^(RFP)IL-17A^(eGFP) or Dominant NegativeIL-10R−Foxp3^(RFP)IL-17A^(eGFP) double reporter mice. FACS-sorted CD4⁺TCRβ⁺Foxp3^(RFP−)CD49b⁺LAG-3⁺ T cells from the small intestine ofanti-CD3 treated mice were transferred i.p. in combination with eT_(H)17cells, or with DNR eT_(H)17 (ratio 1:1) into RAG1^(−/−) mice. Endoscopiccolitis score (FIG. 12B) and change in body weight (FIG. 12C) weremeasured. Each dot represents one mouse. Lines indicate mean±SEM. *P0.05, **P 0.005.

FIG. 13, comprising FIG. 13A through FIG. 13D, depicts the results ofexperiments demonstrating the in vitro regulatory activity of murineCD4⁺CD49b⁺LAG-3⁺ T cells isolated from the spleen of anti-CD3 treatedmice. FIG. 13A: Expression of LAG-3 and CD49b measured on CD4⁺TCRβ⁺Foxp3^(RFP−) in cells isolated from the spleen of anti-CD3 treatedmice (upper panel) and frequencies of CD4⁺IL-10^(eGFP+) T cells (gatedon CD4⁺ TCRβ⁺Foxp3′) in the indicated T cell populations (lower panel)are shown. Representative dot plots from 1 experiment out of 5 areshown. In each experiment cells isolated from 2 to 5 mice were pooled.Percentages of cells in each quadrant are indicated. MFI forIL-10^(eGFP+) T cells in the indicated T cell populations (right panel)are shown. FIG. 13B: Mean±SEM of IL-10^(eGFP+) cell frequencies amongthe indicated T cell populations obtained in 3 independent experimentsis shown. In each experiment 2 to 5 mice were pooled. ***P 0.0005. FIG.13C: The indicated FACS-sorted T cell populations (gated on CD4⁺TCRβ⁺Foxp3^(RFP−)) from the spleen of anti-CD3 treated mice werere-stimulated in vitro with anti-CD3 and anti-CD28 mAbs for cytokineproduction. Mean±SEM of IL-10, IFN-γ, IL-17A, IL-2, IL-4 is shown. Onerepresentative experiment out of 3 is shown. In each experiment cellsisolated from 2 to 5 mice were pooled before the FACS-sorting. Eachexperiment contains at least 2 sample replicates for each population. *P0.05 and **P 0.005. When not indicated differences were notstatistically different. FIG. 13D: The indicate FACS-sorted T cellpopulations (gated on CD4⁺ TCRβ⁺Foxp3^(RFP−)) from the spleen ofanti-CD3 treated mice were tested in suppressive assay in the presenceor absence of anti-IL-10R mAbs. Percentages of suppression mediated bythe indicated T cell populations are reported. *P 0.05, **P 0.005.

FIG. 14, comprising FIG. 14A through FIG. 14E, depicts the results ofexperiments demonstrating that murine CD4⁺CD49b⁺LAG-3⁺ T cells can beisolated from N. brasiliensis infected mice. FIG. 14A: Numbers of totalcells infiltrating the lungs at different time points during theinfection are shown (Mean±SEM). Mice per time points: day 0, n=7; day 5,n=4; day 7, n=5; day 10, n=7; day 16, n=5. FIG. 14B: Frequencies of Ly6Gcells among CD45⁺cells infiltrating the lung at different time pointsduring the infection are shown (Mean±SEM). Mice per time points: day 0,n=3; day 5, n=3; day 7, n=3; day 10, n=3; day 16, n=3. FIG. 14C:Expression of CD49b and LAG-3 measured on CD4⁺TCRβ⁺IL-4^(eGFP+)(T_(H)2); CD4⁺TCRβ⁺Foxp3^(RFP-)IL-17A^(eGFP+)(T_(H)17), CD4⁺TCRβ⁺Foxp3^(RFP+)IL-17^(eGFP−)(Foxp3⁺ Tregs), and CD4⁺TCRβ⁺Foxp3^(RFP−)IL-10^(eGFP+)(Tr1) cells isolated from the lungs ofIL-4^(eGFP), Foxp3^(RFP) IL-17^(eGFP), Foxp3^(RFP) IL-10^(eGFP) reportermice infected with N. brasiliensis. For T_(H)2 and T_(H)17 cells,representative dot plots from 1 experiment out of 3 are shown. ForFoxp3⁺ Tregs cells representative dot plots from 1 experiment out of 4are shown. For Tr1 cells representative dot plots from 1 experiment outof 4 are shown. Percentages of cells in each quadrant are indicated.FIG. 14D: Expression of CD49b and LAG-3 on CD4⁺TCRβ⁺Foxp3⁻IL-17A^(eGFP+)(eT_(H)17) and on CD4⁺TCRβ⁺Foxp3^(RFP−)IFN-γ^(Katushka+)(eT_(H)1) cells isolated from inflamedcolon of RAG1^(−/−) mice transferred with CD4⁺CD45RB^(High) T cellsisolated either from Foxp3^(RFP)IL-17A^(eGFP) cells orFoxp3^(RFP)IFN-γ^(Katushka) double reporter mice. Percentages of cellsin each quadrant are indicated. Representative dot plots from 1experiment out of 3 are shown. FIG. 14E: Mean±SEM of frequencies of theindicated T cell populations obtained in 3-4 independent experiments isshown. In each experiment 2 to 5 mice were pooled. ***P 0.0005. When notindicated differences were not statistically different.

FIG. 15, comprising FIG. 15A through FIG. 15C, depicts the results ofexperiments demonstrating that murine CD4⁺CD49b⁺LAG-3⁺ T cells isolatedfrom N. brasiliensis infected mice expressed high levels of IL-10 andAhR. FIG. 15A: Expression of Il10, Il4, Il13, Gata3, Ahr (relative toHrpt) measured by RT-PCR in the indicated T cell populations isolatedfrom IL-4^(eGFP) and Foxp3^(RFP)IL-10^(eGFP) reporter mice infected withN. brasiliensis. As control CD4⁺ TCRβ+IL-4^(eGFP+)(T_(H)2) and CD4⁺TCRβ⁺Foxp3^(RFP+)(Tregs) isolated from the lung were used. Mean±SEM of 3independent experiments is shown. *P 0.05, **P 0.005 and ***P 0.0005 vs.CD4+CD49b+LAG-3+ T cells. When not indicated differences were notstatistically different. Frequencies of the indicated T cell populations(gated on CD4⁺ TCRβ⁺Foxp3^(RFP−) T cells) accumulated in the lungs (FIG.15B) and in the mediastinal lymph nodes (FIG. 15C) at different timepoints after the N. brasiliensis infection of wild type mice. Mean±SEMis shown. In each time point 2 to 5 mice were tested.

FIG. 16, comprising FIG. 16A through FIG. 16C, depicts the results ofexperiments demonstrating that in vitro differentiated Tr1 cellsco-express CD49b and LAG-3. FIG. 16A: Expression of the indicated genes(normalized to Hprt) in in vitro differentiated T_(H)0, iTregs, T_(H)2,T_(H)17, T_(H)1, and Tr1 cells measured by RT-PCR. T_(H)0 cells wereused as internal control and the expression of each gene in each T cellis normalized to T_(H)0 cells. Mean±SEM of triplicates are shown. ***P0.0005. When not indicated differences were not statistically different.FIG. 16B: Expression of CD49b and LAG-3 in the indicated T cellsdifferentiated in vitro after 4 days of culture is shown. Percentages ofcells in each quadrant are indicated. Representative dot plots from 1experiment out of 3 are shown. FIG. 16C: Mean±SEM of the frequencies ofcells co-expressing CD49b and LAG-3 among the indicated T cellpopulations (n=3 for each group) is shown. *** P 0.0005.

FIG. 17, comprising FIG. 17A and FIG. 17B, depicts the results ofexperiments demonstrating that CD49b and LAG-3 are expressed over timeon in vitro generated Tr1 cells. CD4⁺ T cells were isolated from thespleen of wild type mice and in vitro differentiated in Tr1 cells withIL-27 and TGF-β. FIG. 17A: After 5 days CD4⁺CD49b⁺LAG-3⁺IL-10⁺ Tr1 cellswere FACS sorted and activated in the presence of anti-CD3 and anti-CD28mAbs (upper panel) or anti-CD3, anti-CD28, TGF-β, IL-6 and IL-23 (lowerpanel). The expression of IL-10^(eGFP) and CD49b/LAG-3 was analyzed atthe indicated time points by FACS. FIG. 17B: Sorted Tr1 cells werecultured for 4 days in the presence of anti-CD3, anti-CD28, TGF-β, IL-6and IL-23. The expression of IL-10 among CD49b⁺LAG-3⁺and CD49b″LAG-3^(+/−) is reported.

FIG. 18, comprising FIG. 18A through FIG. 18C, depicts the results ofexperiments demonstrating that upon transfer in vivo, CD49b and LAG-3are expressed on in vitro generated Tr1 cells. CD4⁺ T cells wereisolated from the spleen of wild type mice and in vitro differentiatedin Tr1 cells with IL-27 and TGF-β. After 5 days CD4⁺CD49b⁺LAG-3⁻IL-10⁺Tr1 cells were FACS sorted transferred into RAG-1^(−/−) mice. FIG. 18A:Each mouse was injected i.p. with 10⁵ Tr1 cells and treated as depictedin the cartoon. FIG. 18B-C: The frequency of the indicated populationswas analyzed at the indicated time points by FACS.

FIG. 19, comprising FIG. 19A and FIG. 19B, depicts the results ofexperiments demonstrating that co-expression of CD49b and LAG-3 allowsthe selection of human Tr1 cells in vitro. FIG. 19A: Expression of CD49band LAG-3 in pTr1 and T_(H)0 cell lines. Dot plots from 1 representativedonor out of 7 donors tested is presented. Percentages of cells in eachquadrant are indicated. FIG. 19B: CD49b⁺LAG-3⁺ T cells were FACS-sortedfrom pTr1(DC-10) cells and were tested for their ability to suppress Tcells activated with mDC (responder cells, filled histogram).pTr1(DC-10) cells and CD49b⁺LAG-3⁺ T cells sorted from pTr1(DC-10) cells(CD49b⁺LAG-3⁺pTr1(DC-10)) were used to suppress the proliferation ofautologous CD4+ T cells activated with mDC. As control, T(mDC) cellswere used. Percentages of suppression are indicated. One representativeexperiment out of 3 is shown.

FIG. 20, comprising FIG. 20A and FIG. 20B, depicts the results ofexperiments demonstrating the sensitivity and specificity of theco-expression of CD49b and LAG-3 on human CD4⁺CD45RA″ T cells. EmpiricalReceiver Operating Characteristic (ROC) curves generated by comparingsubjects with persistent mixed chimerism after allogeneic HSCT (PMC,n=11) with healthy donors (HDs, n=23) (FIG. 20A), or with subjects withcomplete chimerism (CC, n=7) (FIG. 20B). Area under the curve (AUC) were0.900 and 0.916, respectively. A threshold of 3.64% for CD49b⁺LAG-3⁺ Tcells gave 81.8% sensitivity and 91.3% specificity when PMC werecompared to HDs. A threshold of 2.765% for CD49b⁺LAG-3⁺ T cells gave 91%sensitivity and 87.5% specificity when PMC were compared to CC.

DETAILED DESCRIPTION

The present invention is based upon the discovery that T regulatory type1 (Tr1) cells express particular cell surface markers that allow fortheir selection, enrichment, isolation, purification and administration.The ability to use the particular markers described herein to select,enrich, isolate, purify and administer Tr1 cells allows for improvedmethods of Tr1 therapies for treating a wide variety of diseases anddisorders.

The invention includes methods of administering Tr1 cells to a subjectin need thereof, to treat or prevent a disease or disorder involving anundesired immune response. Exemplary diseases and disorders that aretreatable or preventable with the Tr1 cell compositions and methods ofthe invention include, but are not limited to, inflammatory diseases anddisorders, autoimmune diseases or disorders, and disorders associatedwith transplantation, such as transplant rejection and graft versus hostdisease.

In certain embodiments, the methods of the invention comprise isolatingT cells which express one or more Tr1 selective markers. In oneembodiment, the method comprises selecting T cells which express one ormore Tr1 marker selected from the group consisting of CD49b, LAG-3, andCD226 (DNAM-1). In some embodiments, the method comprises selecting Tcells that co-express CD49b and LAG-3. In other embodiments, the methodcomprises selecting T cells that co-express CD49b, LAG-3, and CD226. Insome embodiments, the Tr1 cells do not constitutively express highlevels of Foxp3, as compared with the level of Foxp3 on a comparatorcell selected from the group consisting of a CD25bright T cell and aFoxp3+ Treg cell. In some embodiments, the Tr1 cells exhibit IL-10dependent regulatory activity.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

As used herein, to “alleviate” a disease means reducing the severity ofone or more symptoms of the disease.

“Allogeneic” refers to a graft derived from a different animal of thesame species.

“Alloantigen” is an antigen that differs from an antigen expressed bythe recipient.

The term “antibody” as used herein, refers to an immunoglobulinmolecule, which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoactive portions ofintact immunoglobulins. Antibodies are typically tetramers ofimmunoglobulin molecules. The antibodies in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, Fv, Fab and F(ab).sub.2, as well assingle chain antibodies and humanized antibodies (Harlow et al., 1988;Houston et al., 1988; Bird et al., 1988).

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample. Such a biological sample can include, but isnot limited to a tissue sample, a tumor sample, a cell or a biologicalfluid.

“An antigen presenting cell” (APC) is a cell that is capable ofactivating T cells, and includes, but is not limited to,monocytes/macrophages, B cells and dendritic cells (DCs).

The term “dendritic cell” or “DC” refers to any member of a diversepopulation of morphologically similar cell types found in lymphoid ornon-lymphoid tissues. These cells are characterized by their distinctivemorphology, high levels of surface MHC-class II expression. DCs can beisolated from a number of tissue sources. DCs have a high capacity forsensitizing MHC-restricted T cells and are very effective at presentingantigens to T cells in situ. The antigens may be self-antigens that areexpressed during T cell development and tolerance, and foreign antigensthat are present during normal immune processes.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the mammal.

As used herein, by “combination therapy” is meant that a first agent isadministered in conjunction with another agent. “In conjunction with”refers to administration of one treatment modality in addition toanother treatment modality. As such, “in conjunction with” refers toadministration of one treatment modality before, during, or afterdelivery of the other treatment modality to the individual. Suchcombinations are considered to be part of a single treatment regimen orregime.

As used herein, the term “concurrent administration” means that theadministration of the first therapy and that of a second therapy in acombination therapy overlap with each other.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated,then the animal's health continues to deteriorate. In contrast, a“disorder” in an animal is a state of health in which the animal is ableto maintain homeostasis, but in which the animal's state of health isless favorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

The term “DNA” as used herein is defined as deoxyribonucleic acid.

“Donor antigen” refers to an antigen expressed by the donor tissue to betransplanted into the recipient.

“Recipient antigen” refers to an antigen expressed by the recipient.

As used herein, an “effector cell” refers to a cell which mediates animmune response against an antigen. An example of an effector cellincludes, but is not limited to a T cell and a B cell.

As used herein, the term “immune response” includes T cell mediatedand/or B-cell mediated immune responses. Exemplary immune responsesinclude T cell responses, e.g., cytokine production and cellularcytotoxicity, and B cell responses, e.g., antibody production. Inaddition, the term immune response includes immune responses that areindirectly affected by T cell activation, e.g., antibody production(humoral responses) and activation of cytokine responsive cells, e.g.,macrophages. Immune cells involved in the immune response includelymphocytes, such as B cells and T cells (CD4+, CD8+, Th1 and Th2cells); antigen presenting cells (e.g., professional antigen presentingcells such as dendritic cells, macrophages, B lymphocytes, Langerhanscells, and non-professional antigen presenting cells such askeratinocytes, endothelial cells, astrocytes, fibroblasts,oligodendrocytes); natural killer cells; myeloid cells, such asmacrophages, eosinophils, mast cells, basophils, and granulocytes.

“Mixed lymphocyte reaction,” “mixed lymphocyte culture,” “MLR,” and“MLC” are used interchangeably to refer to a mixture comprising aminimum of two different cell populations that are allotypicallydifferent. At least one of the allotypically different cells is alymphocyte. The cells are cultured together for a time and undersuitable conditions to result in the stimulation of the lymphocytes,including for example, Tr1 cells. A frequent objective of an MLC is toprovide allogeneic stimulation, such as may initiate proliferation ofthe Tr1 cells; but unless indicated, proliferation during the culture isnot required. In the proper context, these terms may alternatively referto a mixture of cells derived from such a culture.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

By the term “effective amount,” as used herein, is meant an amount thatwhen administered to a mammal, causes a detectable level of immunesuppression or tolerance compared to the immune response detected in theabsence of the composition of the invention. The immune response can bereadily assessed by a plethora of art-recognized methods. The skilledartisan would understand that the amount of the composition administeredherein varies and can be readily determined based on a number of factorssuch as the disease or condition being treated, the age and health andphysical condition of the mammal being treated, the severity of thedisease, the particular compound being administered, and the like.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “epitope” as used herein is a portion of an antigen that canelicit an immune response, including B and/or T cell responses. Anantigen can have one or more epitopes.

Most antigens have many epitopes; i.e., they are multivalent. In someexamples, an epitope is roughly about 10 amino acids and/or sugars insize. Preferably, the epitope is about 4-18 amino acids, more preferablyabout 5-16 amino acids, and even more most preferably 6-14 amino acids,more preferably about 7-12, and most preferably about 8-10 amino acids.One skilled in the art understands that in some circumstances, thethree-dimensional structure, rather than the specific linear sequence ofthe molecule, is the main criterion of antigenic specificity andtherefore distinguishes one epitope from another.

The term “expression” as used herein is defined as the transcriptionand/or translation of a nucleotide sequence.

The term “expression vector” as used herein refers to a vectorcontaining a nucleic acid sequence coding for at least part of a geneproduct capable of being transcribed. In some cases, RNA molecules arethen translated into a protein, polypeptide, or peptide. In other cases,these sequences are not translated, for example, in the production ofantisense molecules, siRNA, ribozymes, and the like. Expression vectorscan contain a variety of control sequences, which refer to nucleic acidsequences necessary for the transcription and possibly translation of anoperatively linked coding sequence in a particular host organism. Inaddition to control sequences that govern transcription and translation,vectors and expression vectors may contain nucleic acid sequences thatserve other functions as well.

The term “helper T cell” as used herein is defined as an effector T cellwhose primary function is to promote the activation and functions ofother B and T lymphocytes and or macrophages. Many helper T cells areCD4 T-cells.

The term “heterologous” as used herein is defined as DNA or RNAsequences or proteins that are derived from the different species.

As used herein, “homology” is used synonymously with “identity.”

The term “immunoglobulin” or “Ig,” as used herein is defined as a classof proteins, which function as antibodies. The five members included inthis class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is theprimary antibody that is present in body secretions, such as saliva,tears, breast milk, gastrointestinal secretions and mucus secretions ofthe respiratory and genitourinary tracts. IgG is the most commoncirculating antibody. IgM is the main immunoglobulin produced in theprimary immune response in most mammals. It is the most efficientimmunoglobulin in agglutination, complement fixation, and other antibodyresponses, and is important in defense against bacteria and viruses. IgDis the immunoglobulin that has no known antibody function, but may serveas an antigen receptor. IgE is the immunoglobulin that mediatesimmediate hypersensitivity by causing release of mediators from mastcells and basophils upon exposure to allergen.

The term “immunostimulatory” is used herein to refer to increasing atleast one parameter of an immune response.

The term “immunosuppressive” is used herein to refer to reducing atleast one parameter of an immune response.

“Tr1 differentiation” as used herein refers to any event which resultsin a detectable increase in the phenotype and/or genotype characteristicof Tr1 cells. For example, a phenotype and/or genotype characteristic ofTr1 cells is the co-expression of CD49b and LAG-3. Another phenotypeand/or genotype characteristic of Tr1 cells is immunosuppression.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR, and thelike, and by synthetic means.

The term “polypeptide” as used herein is defined as a chain of aminoacid residues, usually having a defined sequence. As used herein theterm polypeptide is mutually inclusive of the terms “peptide” and“protein.”

The term “self-antigen” as used herein is defined as an antigen that isexpressed by a host cell or tissue. Self-antigens may be tumor antigens,but in certain embodiments, are expressed in both normal and tumorcells. A skilled artisan would readily understand that a self-antigenmay be overexpressed in a cell.

As used herein, “specifically binds” refers to the fact that a firstcompound binds preferentially with a second compound and does not bindin a significant amount to other compounds present in the sample.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cells that have been separated from the cells with which theyare naturally associated in their natural state. In some embodiments,the cells are cultured in vitro. In other embodiments, the cells are notcultured in vitro.

As the term is used herein, “substantially separated from” or“substantially separating” refers to the characteristic of a populationof first substances being removed from the proximity of a population ofsecond substances, wherein the population of first substances is notnecessarily devoid of the second substance, and the population of secondsubstances is not necessarily devoid of the first substance. However, apopulation of first substances that is “substantially separated from” apopulation of second substances has a measurably lower content of secondsubstances as compared to the non-separated mixture of first and secondsubstances. In some examples, the first substance is a particular typeof cell identifiable by is expression of cell surface markers.

A “population” is used herein to refer to a group of cells having asubstantially similar phenotypic characteristic.

“Transplant” refers to a donor tissue, organ or cell, to betransplanted. An example of a transplant may include but is not limitedto skin cells or tissue, hematopoietic cells, bone marrow, and solidorgans such as heart, pancreas, kidney, lung and liver.

The term “T-cell” as used herein is defined as a thymus-derived cellthat participates in a variety of cell-mediated immune reactions.

The term “B-cell” as used herein is defined as a cell derived from thebone marrow and/or spleen. B cells can develop into plasma cells whichproduce antibodies.

As used herein, a “therapeutically effective amount” is the amount of atherapeutic composition sufficient to provide a beneficial effect to amammal to which the composition is administered.

As used herein, “treating” refers to the reduction, alleviation orelimination, of at least one sign or symptom of a disease or disorderwhich is being treated, e.g. alleviation of immune dysfunction oravoidance of transplant rejection, relative to the symptoms prior totreatment. As used herein “treating” or “treatment” includes boththerapeutic and prophylactic treatments.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

“Xenogeneic” refers to a graft derived from an animal of a differentspecies.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

The present invention is based upon the finding that T regulatory type 1(Tr1) cells express specific cell surface markers that allow for theirselection, enrichment, isolation, and purification. While certainmethods of generating Tr1 cells are known in the art, there has, untilnow, yet to be a method of producing an enriched population of Tr1 cellsfor use in research and clinical therapeutic methods. The ability to usethe specific markers described herein to select, enrich, isolate, andpurify Tr1 cells allows for improved methods of Tr1 therapies fortreating a wide variety of diseases and disorders. For example, it isdemonstrated herein that Tr1 cells selected, enriched, isolated, andpurified by the methods of the invention exhibit immunosuppressiveactivities both in vitro and in vivo.

The invention includes methods of administering Tr1 cells to a subjectin need thereof, to treat or prevent a disease or disorder involving anundesired immune response. Exemplary diseases and disorders that aretreatable or preventable with the Tr1 cell compositions and methods ofthe invention include, but are not limited to, inflammatory diseases anddisorders, autoimmune diseases or disorders, and disorders associatedwith transplantation, such as transplant rejection and graft versus hostdisease. Examples of autoimmune and inflammatory diseases and disorderstreatable or preventable with the Tr1 cell compositions and methods ofthe invention include, but are not limited to, acute and chronicdiseases and disorders such as allergy, asthma, inflammatory boweldisease, autoimmune entheropathy, Addision's disease, alopecia areata,ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis,Crohn's disease, diabetes mellitus, dystrophic epidermolysis bullosa,epididymitis, glomerulonephritis, Graves' disease, Guillain-Barrsyndrome, Hashimoto's disease, hemolytic anemia, systemic lupuserythematosus, multiple sclerosis, myasthenia gravis, pemphigusvulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis,scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis,vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerativecolitis. In certain embodiments, the Tr1 cell compositions and methodsof the invention are used to treat subjects who have received atransplant, such as a hematopoietic cell transplant, a stem celltransplant, a bone marrow transplant, cord blood transplant, an organand cell transplant, a blood transfusion, and the like.

In certain embodiments, the methods of the invention comprise selectingT cells which express one or more Tr1 selective markers. In oneembodiment, the method comprises selecting T cells which express one ormore Tr1 marker selected from the group consisting of CD49b, LAG-3, andCD226 (DNAM-1). In some embodiments, the method comprises selecting Tcells that co-express CD49b and LAG-3. In other embodiments, the methodcomprises selecting T cells that co-express CD49b, LAG-3, and CD226. Insome embodiments, the method comprises selected T cells that expressCD49b, LAG-3, and an elevated level of CD226, as compared with the levelof CD226 on a comparator cell population, such as CD49b⁻LAG-3⁻ T cells,or T_(H)0 cells. In some embodiments, the Tr1 cells do notconstitutively express high levels of Foxp3, as compared with the levelof Foxp3 on a comparator cell selected from the group consisting of aCD25bright T cell and a Foxp3+Treg cell. In some embodiments, the Tr1cells exhibit IL-10 dependent regulatory activity. In certainembodiments, the method comprises selecting T cells which express one ormore Tr1 markers after the T cells are activated.

In one embodiment, the invention comprises detecting the level of Tr1cells in a subject by detecting the absolute number, or the relativeamount, of T cells which express Tr1 markers in a sample obtained fromthe subject. In one embodiment, the method comprises detecting T cellswhich express one or more Tr1 markers selected from the group consistingof CD49b, LAG-3, and CD226. In some embodiments, the Tr1 cells do notconstitutively express high levels of Foxp3, as compared with the levelof Foxp3 on a comparator cell selected from the group consisting of aCD25bright T cell and a Foxp3+Treg cell. In some embodiments, the Tr1cells exhibit IL-10 dependent regulatory activity. The method can beused to determine if the subject is tolerized or tolerant to atransplantation therapy, including, but not limited to a hematopoieticcell transplantation, such as a hematopoietic stem cell transplantation(HSCT). In one embodiment, the method can be used to monitor theabsolute number or relative amount of Tr1 cells in a subject over time,thereby allowing for the prediction of the risk of an adverse immuneresponse.

Tr1 Cell Differentiation

The invention includes methods of and compositions for converting ordifferentiating non-regulatory T cells into Tr1 cells. In oneembodiment, the method comprises converting non-regulatory T cells intoTr1 cells that express at least one marker selected from the groupconsisting of CD49b, LAG-3, and CD226 (DNAM-1). In some embodiments, themethod comprises converting non-regulatory T cells into Tr1 cells thatco-express CD49b and LAG-3. In other embodiments, the method comprisesconverting non-regulatory T cells into Tr1 cells that co-express CD49b,LAG-3, and CD226. The method comprises converting non-regulatory T cellsinto Tr1 cells that do not constitutively express high levels of Foxp3,as compared with the level of Foxp3 on a comparator cell selected fromthe group consisting of a CD25bright T cell and a Foxp3+ Treg cell. Insome embodiments, the method comprises converting non-regulatory T cellsinto Tr1 cells that exhibit IL-10 dependent regulatory activity.

In some embodiments, the method of differentiating cells into Tr1 cellsincludes the step of obtaining non-regulatory T cells of a subject. Insome embodiment, the non-regulatory T cells of the subject are CD4⁺ Tcells. In some embodiments, the non-regulatory T cells the subject areCD4⁺CD25⁻ T cells. In some embodiments, the subject is a mammal, such asa human or a mouse. In some embodiments, the method of differentiatingcells into Tr1 cells includes the step of culturing the non-regulatory Tcells of the subject in the presence of feeder cells. In someembodiments, the feeder cells are L cells. In some embodiments, thefeeder cells are transfected with at least one of CD32, CD80, and CD58.In some embodiments, the feeder cells are transfected with at least oneof hCD32, hCD80, and hCD58. In some embodiments, the method ofdifferentiating cells into Tr1 cells includes the step of culturing thenon-regulatory T cells of the subject in the presence of anti-CD3 mAb.In some embodiments, the method of differentiating cells into Tr1 cellsincludes the step of culturing the non-regulatory T cells of the subjectin the presence of IL-2, such as rhIL-2. In some embodiments, the methodof differentiating cells into Tr1 cells includes the step of culturingthe non-regulatory T cells of the subject in the presence of IL-15, suchas rhIL-15. In some embodiments, the differentiated Tr1 cells arepolarized. In some embodiments, the differentiated Tr1 cells arepolarized by culturing the differentiated Tr1 cells in the presence ofat least one of IL-10, such as rhIL-10, and IFNα-2b, such as rhIFNα-2b.

The invention also provides methods and compositions for ex vivoconversion and expansion of Tr1 cells from non-Tr1 cells. The expansionmethods for Tr1 cells can include the use of a bead- or cell-basedartificial antigen-presenting cell. However, any method in the art canbe used to expand the Tr1.

The present invention provides a method of large-scale conversion andexpansion of Tr1 that addresses the low numbers of natural Tr1 cellsthat can be isolated and expanded. Thus, the methods and compositions ofthe invention are useful for therapeutic purposes, for example, in theprevention and treatment of immune-based disorders and in the preventionand treatment of allograft rejection.

Tr1 Cell Isolation and Expansion

Tr1 cells suppress immune responses and play an important role inimmunotherapy against inflammation and autoimmune disease and contributeto transplantation tolerance. Some in vivo uses require expansionprocesses to generate sufficient numbers of Tr1 cells for in vivotherapeutic use.

The present invention provides a method of generating an enrichedpopulation of immunosuppressive Tr1 cells from the abundant CD4⁺ T cellpopulation. The various embodiments, the majority of the cells of theenriched population of Tr1 cells express the cell surface markers CD4,and CD49b, and LAG-3. In some embodiments, greater than 90% the cells ofthe enriched population of Tr1 cells express the cell surface markersCD4, and CD49b, and LAG-3. In some embodiments, greater than 95% thecells of the enriched population of Tr1 cells express the cell surfacemarkers CD4, and CD49b, and LAG-3. In some embodiments, greater than 98%the cells of the enriched population of Tr1 cells express the cellsurface markers CD4, and CD49b, and LAG-3. In some embodiments, greaterthan 99% the cells of the enriched population of Tr1 cells express thecell surface markers CD4, and CD49b, and LAG-3. In some embodiments,greater than 99.5% the cells of the enriched population of Tr1 cellsexpress the cell surface markers CD4, and CD49b, and LAG-3.

This method allows for the generation of Tr1 cells in sufficient numbersfor in vivo infusions. The method can be used both for generating Tr1cells for research purposes as well as for clinical use byadministration to a subject in need thereof.

The present invention provides a method of generating a population ofimmunosuppressive Tr1 cells from the abundant CD4⁺ T cell population.This method allows for the generation of Tr1 cells in sufficient numbersfor in vivo infusions. The method can be used both for generating Tr1cells for research purposes as well as for clinical use byadministration to a subject in need thereof.

In some embodiments, the invention provides methods of selecting orisolating the cells so identified. In some embodiments, CD4⁺ T cells areobtained from blood (e.g., isolated from PBMC), bone marrow, cord blood,lymphoid tissue, thymus, spleen, or any tissues/organ sample ofinterest, including, but not limited the pancreas, eye, heart, liver,nerves, intestine, skin, muscle, and joints.

The cells bearing the desired markers (e.g., CD49b and LAG-3) can beisolated, for instance, by the use of labeled antibodies or ligands withFACS or magnetic particles/bead technologies as known to one of ordinaryskill in the art. Accordingly, in some embodiments, the inventionprovides a method of generating an enriched population ofimmunosuppressive Tr1 cells which are substantially CD4⁺CD49b⁺LAG-3⁺ byobtaining a biological sample that also comprises non-Tr1 cells,including, but not limited to, CD4⁺, CD4⁺CD25⁻, CD4⁺CD25⁻CD45RA⁺cells,and converting or differentiating the non-Tr1 cells into Tr1 cells.

To enhance the enrichment of Tr1 cells, positive selection for CD49band/or LAG-3 may be combined with negative selection against cellsurface makers specific to non-Tr1 cell types, including, by way ofnon-limiting examples, CD8, CD11b, CD16, CD19, CD36 and CD56.

Sources of T cells and methods of isolating particular T cellpopulations (e.g., CD4⁺cells) which can be converted or differentiatedby culturing according to the methods of the present invention are wellknown and described in the literature. Thus for example T cells mayconveniently be isolated from the blood e.g. from a peripheral bloodmononuclear cell (PBMC) population isolated from blood, or from otherblood-derived preparations such as leukopheresis products or from bonemarrow, lymph, thymus, spleen or umbilical cord. T cell populations maybe derived from any appropriate source, including human or animalsources.

The invention includes converting or differentiating non-Tr1 cells, ormixed populations of Tr1 cells and non-Tr1 cells, in the presence of abead- or cell-based artificial antigen-presenting cell system.Regardless of the system used for cellular expansion, the cells can beexpanded prior to, simultaneously with, and/or subsequent to Tr1conversion. For example, the cells can be expanded using a bead- orcell-based artificial antigen-presenting cell system before the initialTr1 conversion stage. Alternatively, the cells can be expanded using abead- or cell-based artificial antigen-presenting cell system after theinitial Tr1 conversion stage but before the selective outgrowth stagethat favors proliferation of Tr1s. Alternatively, the cells can beexpanded using a bead- or cell-based artificial antigen-presenting cellsystem after the outgrowth stage but before the imprinting stage.Alternatively, the cells can be expanded using a bead- or cell-basedartificial antigen-presenting cell system after the imprinting state.

Special cell-sized beads (e.g., magnetic iron-dextran beads) can be usedthat are coated with antibodies, such as anti-CD3 and/or anti-CD28. Theuse of anti-CD3 and/or anti-CD28 beads induced robust proliferation ofcells. As a non-limiting example, a 3:1 bead:T cell ratio expands andpreserves Tr1 function at a desirable level. The ratios of antibodies toCD3 and/or CD28 can be adjusted for optimal results. The beads caneasily be removed by passing the cultured cells through a magneticcolumn. As an added advantage, the culture-expanded Tr1 retain potentfunctional suppressor activity.

The culture-expanded Tr1 of the present invention are capable ofsuppressing an MLR, with, by way of example, primary CD4⁺cells orcultured CD4⁺CD25⁻cells as responding T cells. In one embodiment theconverted and expanded Tr1 cells inhibit the autologous proliferation ofperipheral blood cells. In another embodiment, the converted andexpanded Tr1 cells block or prevent GVHD, or inhibit or reverse thedisease if already in progress. In yet another embodiment, the convertedand expanded Tr1 cells are introduced into a different host; whereas inyet another embodiment, the Tr1 cells are established as a cell line forcontinuous therapeutic use. Preferably, the host is a human host and theculture-expanded Tr1 cells are human, although animals, including animalmodels for human disease states, are also included in this invention andtherapeutic treatments of such animals are contemplated herein.

Following Tr1 conversion or differentiation using the methods of theinvention, Tr1 cells can be expanded under appropriate conditions forgrowth of the Tr1 cells. Growth is allowed to progress for a time periodselected according to the final number of T cells required and the rateof expansion of the cells. Passaging of the cells may be undertakenduring this period. Such a time period is normally between 3 and 10 daysbut can be as long as 14 to 20 days or even longer providing theviability and continued proliferation of the T cells is maintained.

Therapeutic Application

The invention includes methods of administering Tr1 cells to a subjectin need thereof, for the treatment or prevention of a disease ordisorder, such as an inflammatory disease or disorder, an autoimmunedisease or disorder, or transplantation rejection. The ex vivoculture-converted and culture-expanded Tr1 cells, with or withoutnaturally occurring Tr1 cells, can be introduced to the host subject orto another subject by any number of approaches. In some embodiments,they are injected intravenously. Optionally, the host subject may betreated with agents to promote the in vivo function and survival of theTr1 cells. Of course, the culture-expanded Tr1 may also be introduced ina variety of pharmaceutical formulations. These may contain suchnormally employed additives as binders, fillers, carriers,preservatives, stabilizing agents, emulsifiers, and buffers. Suitablediluents and excipients are, for example, water, saline, and dextrose,as utilized in the methods described herein. The administration of Tr1cells to a subject before, during or after onset of the disease ordisorder, serves to diminish the frequency or severity of the signs orsymptoms of the disease or disorder experienced by the subject.

In various embodiments, the cells can be converted directly afterharvest or the cells can be stored (e.g., by freezing) prior to theirexpansion, or the cells can be stored (e.g., by freezing) afterexpansion and prior to their therapeutic administration. In variousembodiments, the Tr1 cells of the invention can be administered alone,or the Tr1 cells of the invention can be administered in combinationwith a known immunosuppressive therapy.

The methods of the invention thus provide for achieving animmunosuppressive effect in a subject, i.e., a method of preventing ordiminishing an immune response. The disease or disorder typified by anaberrant immune response may be an inflammatory or autoimmune disease ordisorder, such as allergy, asthma, inflammatory bowel disease,autoimmune entheropathy, Addision's disease, alopecia areata, ankylosingspondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn'sdisease, diabetes mellitus, dystrophic epidermolysis bullosa,epididymitis, glomerulonephritis, Graves' disease, Guillain-Barrsyndrome, Hashimoto's disease, hemolytic anemia, systemic lupuserythematosus, multiple sclerosis, myasthenia gravis, pemphigusvulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis,scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis,vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerativecolitis.

In certain embodiments, the Tr1 cell compositions and methods of theinvention are used to prevent or treat with an inflammatory disease ordisorder, or an autoimmune disease or disorder, in a subject in needthereof. Non-limiting examples of inflammatory and autoimmune diseasesand disorders preventable or treatable with the compositions and methodsof the invention, include but are not limited to, allergy, asthma,inflammatory bowel disease, autoimmune entheropathy, Addision's disease,alopecia areata, ankylosing spondylitis, autoimmune hepatitis,autoimmune parotitis, Crohn's disease, diabetes mellitus, dystrophicepidermolysis bullosa, epididymitis, glomerulonephritis, Graves'disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia,systemic lupus erythematosus, multiple sclerosis, myasthenia gravis,pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis,sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies,thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, andulcerative colitis.

In other embodiments, the Tr1 cell compositions and methods of theinvention are used to treat subjects who have received a transplant,such as a hematopoietic cell transplant, a stem cell transplant, a bonemarrow transplant, an organ or cell transplant, a blood transfusion, andthe like. Conditions in which immune suppression would be advantageousinclude conditions in which a normal or an activated immune response isdisadvantageous to the mammal, e.g. allotransplantation of cells ortissues, to avoid rejection, or in fertility treatments in whichinappropriate immune responses have been implicated in failure toconceive and miscarriage. The use of such cells before, during, or aftertransplantation avoids extensive chronic graft versus host disease whichmay occur in post-transplant patients. The cells may be convertedimmediately after harvest or stored (e.g., by freezing) prior toexpansion or after expansion and prior to their therapeutic use. Thetherapies may be conducted in conjunction with known immunosuppressivetherapies.

The methods of the present invention are particularly useful for humans,but may also be practiced on veterinary subjects. An “individual,”“subject,” “patient” or “host” referred to herein is a vertebrate,preferably a mammal. More preferably, such individual is a human and theculture-expanded cells are human, although animals, including animalmodels for human disease states, are also included in this invention andtherapeutic treatments of such animals are contemplated herein. Suchanimal models can be used to test and adjust the compositions andmethods of this invention, if desired. Certain models involve injectingin-bred animals with established cell populations. Also useful arechimeric animal models, described in U.S. Pat. Nos. 5,663,481, 5,602,305and 5,476,993; EP application 379,554; and International Appl. WO91/01760. Non-human mammals include, but are not limited to, veterinaryor farm animals, sport animals, and pets. Accordingly, as opposed toanimal models, such animals may be undergoing selected therapeutictreatments.

The present invention encompasses a method of reducing and/oreliminating an immune response in a subject with an inflammatory orautoimmune disease or disorder by administering to the subject an amountof Tr1 cells effective to reduce or inhibit an immune response in thesubject. The Tr1 cells can be administered to the subject, before,during, or after onset of the disease or disorder. Non-limiting examplesof inflammatory and autoimmune diseases and disorders treatable with thecompositions and methods of the invention, include but are not limitedto, allergy, asthma, inflammatory bowel disease, autoimmuneentheropathy, Addision's disease, alopecia areata, ankylosingspondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn'sdisease, diabetes mellitus, dystrophic epidermolysis bullosa,epididymitis, glomerulonephritis, Graves' disease, Guillain-Barrsyndrome, Hashimoto's disease, hemolytic anemia, systemic lupuserythematosus, multiple sclerosis, myasthenia gravis, pemphigusvulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis,scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis,vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerativecolitis.

The present invention encompasses a method of reducing and/oreliminating an immune response to a transplant in a recipient byadministering to the recipient of the transplant an amount of Tr1 cellseffective to reduce or inhibit host rejection of the transplant. The Tr1cells can be administered to the transplant patient, before transplant,during transplant, or after the transplant has occurred. Without wishingto be bound to any particular theory, the Tr1 cells that areadministered to the recipient of the transplant inhibit the activationand proliferation of the recipient's T cells, or induce tolerance. Thetransplant can include a donor tissue, organ or cell. An example of atransplant may include but is not limited to skin cells or tissue,hematopoietic cells, bone marrow, pancreatic islets, and solid organssuch as heart, pancreas, kidney, lung and liver.

In one embodiment, the method of the invention is a method of inhibitinga T cell mediated immune response, by contacting at least one T cellwith an effective amount of CD4+CD49+LAG-3+ Tr1 cells. In oneembodiment, the T cell mediated immune response inhibited by the methodsof the invention is an effector T cell activity. In another embodiment,the T cell mediated immune response inhibited by the methods of theinvention is cytotoxic T-lymphocyte (CTL) activity.

In another embodiment, the method of the invention is a method ofinhibiting at least one alloreactive T cell, by contacting the at leastone alloreactive T cell with an effective amount of CD4+CD49+LAG-3+ Tr1cells.

In one embodiment, the method of the invention is a method of generatingan immunomodulatory effect in a subject having an alloreactive response,inflammatory response, or autoimmune response, the method comprisingadministering to said subject an effective amount of CD4+CD49+LAG-3+ Tr1cells.

In another embodiment, the method of the invention is a method ofpreventing an alloreactive response, inflammatory response, orautoimmune response in a subject, said method comprising administeringto the subject, prior to onset of the alloreactive response,inflammatory response, or autoimmune response, an effective amount ofCD4+CD49+LAG-3+ Tr1 cells to prevent the response.

Based upon the disclosure provided herein, Tr1 cells can be obtainedfrom any source, for example, from the tissue donor, the transplantrecipient or an otherwise unrelated source (a different individual orspecies altogether). The Tr1 cells may be autologous with respect to theT cells (obtained from the same host) or allogeneic with respect to theT cells. In the case where the Tr1 cells are allogeneic, the Tr1 cellsmay be autologous with respect to the transplant to which the T cellsare responding to, or the Tr1 cells may be obtained from a mammal thatis allogeneic with respect to both the source of the T cells and thesource of the transplant to which the T cells are responding to. Inaddition, the Tr1 cells may be xenogeneic to the T cells (obtained froman animal of a different species), for example mouse Tr1 cells may beused to suppress activation and proliferation of human T cells.

Another aspect of the present invention encompasses the route ofadministering Tr1 cells to the subject. Tr1 cells can be administered bya route that is suitable under the circumstances. Tr1 cells can beadministered systemically, i.e., parenterally, by intravenous injectionor can be targeted to a particular tissue or organ, such as bone marrow.Tr1 can be administered via a subcutaneous implantation of cells or byinjection of the cells into connective tissue, for example, muscle.

Tr1s cells can be suspended in an appropriate diluent, at aconcentration of about 5×10⁶ cells/ml. Suitable excipients for injectionsolutions are those that are biologically and physiologically compatiblewith the Tr1s and with the recipient, such as buffered saline solutionor other suitable excipients. The composition for administration can beformulated, produced and stored according to standard methods complyingwith proper sterility and stability.

The dosage of the Tr1 cells varies within wide limits and may beadjusted to the subject's requirements in each particular case. Thenumber of cells used depends on the weight and condition of therecipient, the number and/or frequency of administrations, and othervariables known to those of skill in the art.

In various embodiments, between about 10⁵ and about 10¹³ Tr1 cells per100 kg body weight can be administered to the subject. In someembodiments, between about 1.5×10⁶ and about 1.5×10¹² cells areadministered per 100 kg body weight. In some embodiments, between about1×10⁹ and about 5×10¹¹ cells are administered per 100 kg body weight. Insome embodiments, between about 4×10⁹ and about 2×10″ cells areadministered per 100 kg body weight. In some embodiments, between about5×10⁸ cells and about 1×10¹⁰ cells are administered per 100 kg bodyweight.

In another embodiment of the present invention, Tr1 cells areadministered to the recipient prior to, contemporaneously with, or aftera transplant to reduce and/or eliminate host rejection of thetransplant. While not wishing to be bound to any particular theory, Tr1scan be used to condition a recipient's immune system to the transplantby administering Tr1s to the recipient, prior to, at the same time as,or following transplantation of the transplant, in an amount effectiveto reduce, inhibit or eliminate an immune response against thetransplant by the recipient's T cells. The Tr1 cells affect the T cellsof the recipient such that the T cell response is reduced, inhibited oreliminated when presented with the transplant. Thus, host rejection ofthe transplant may be avoided, or the severity thereof reduced, byadministering Tr1 cells to the recipient, prior to, at the same time as,or following transplantation.

In another embodiment of the present invention, Tr1 cells areadministered to the patient prior to, contemporaneously with, or afterthe onset of inflammatory or autoimmune diseases to prevent and/orre-establish tolerance. While not wishing to be bound to any particulartheory, Tr1s can be used to condition a patient's immune system byadministering Tr1s to the patient, prior to, at the same time as, orfollowing disease onset, in an amount effective to prevent, reduce,inhibit or eliminate an immune response by the patient's T cells. TheTr1 cells affect the T cells of the patients such that the T cellresponse is prevented, reduced, inhibited or eliminated.

Further, the present invention comprises a method of treating a patientwho is undergoing an adverse immune response to a transplant byadministering Tr1 cells to the patient in an amount effective to reduce,inhibit or eliminate the immune response to the transplant, also knownas host rejection of the transplant.

The present invention includes a method of using Tr1 cells as a therapyto inhibit graft versus host disease or graft rejection followingtransplantation. Accordingly, the present invention encompasses a methodof contacting a donor transplant, for example a donor tissue, organ orcell, with Tr1 cells prior to, during, or after transplantation of thetransplant into a recipient. The Tr1 cells serve to ameliorate, inhibitor reduce an adverse response by the donor transplant against therecipient.

As discussed elsewhere herein, Tr1 cells can be obtained from anysource, for example, from the tissue donor, the transplant recipient oran otherwise unrelated source (a different individual or speciesaltogether) for the use of eliminating or reducing an unwanted immuneresponse by a transplant against a recipient of the transplant.Accordingly, Tr1 cells can be autologous, allogeneic or xenogeneic tothe tissue donor, the transplant recipient or an otherwise unrelatedsource.

In an embodiment of the present invention, the transplant is exposed toTr1 cells prior, at the same time, or after transplantation of thetransplant into the recipient. In this situation, an immune responseagainst the transplant caused by any alloreactive recipient cells wouldbe suppressed by the Tr1 cells present in the transplant. The Tr1 cellsare allogeneic to the recipient and may be derived from the donor orfrom a source other than the donor or recipient. In some cases, Tr1cells autologous to the recipient may be used to suppress an immuneresponse against the transplant. In another case, the Tr1 cells may bexenogeneic to the recipient, for example mouse or rat Tr1 cells can beused to suppress an immune response in a human. However, it ispreferable to use human Tr1 cells in the present invention.

In another embodiment of the present invention, the donor transplant canbe “preconditioned” or “pretreated” by contacting the transplant priorto transplantation into the recipient with Tr1 cells in order to reducethe immunogenicity of the transplant against the recipient, therebyreducing and/or preventing graft versus host disease or graft rejection.For example, the transplant can be contacted with cells or a tissue fromthe recipient prior to transplantation in order to activate T cells thatmay be associated with the transplant. Following the treatment of thetransplant with cells or a tissue from the recipient, the cells ortissue may be removed from the transplant. The treated transplant isthen further contacted with Tr1 cells in order to reduce, inhibit oreliminate the activity of the T cells that were activated by thetreatment of the cells or tissue from the recipient. Following thistreatment of the transplant with Tr1 cells, the Tr1 cells may be removedfrom the transplant prior to transplantation into the recipient.However, some Tr1 cells may adhere to the transplant, and therefore, maybe introduced to the recipient with the transplant. In this situation,the Tr1 cells introduced into the recipient can suppress an immuneresponse against the recipient caused by any cell associated with thetransplant. Without wishing to be bound to any particular theory, thetreatment of the transplant with Tr1 cells prior to transplantation ofthe transplant into the recipient serves to reduce, inhibit or eliminatethe activity of the activated T cells, thereby preventing restimulation,or inducing hyporesponsiveness of the T cells to subsequent antigenicstimulation from a tissue and/or cells from the recipient. One skilledin the art would understand based upon the present disclosure, thatpreconditioning or pretreatment of the transplant prior totransplantation may reduce or eliminate the graft versus host response.

In the context of umbilical cord blood, bone marrow or peripheral bloodstem cell (hematopoietic stem cell) transplantation, attack of the hostby the graft can be reduced, inhibited or eliminated by preconditioningthe donor marrow by using the pretreatment methods disclosed herein inorder to reduce the immunogenicity of the graft against the recipient.As described elsewhere herein, a donor hematopoietic stem and progenitorcell source can be pretreated with Tr1 cells from any source, preferablywith recipient Tr1 cells in vitro prior to the transplantation of thedonor marrow into the recipient. In a preferred embodiment, the donormarrow is first exposed to recipient tissue or cells and then treatedwith Tr1 cells. Although not wishing to be bound to any particulartheory, it is believed that the initial contact of the donorhematopoietic stein and progenitor cell source with recipient tissue orcells function to activate the T cells in the donor marrow. Treatment ofthe donor marrow with the Tr1 cells induces hyporesponsiveness orprevents restimulation of T cells to subsequent antigenic stimulation,thereby reducing, inhibiting or eliminating an adverse effect induced bythe donor marrow on the recipient.

In an embodiment of the present invention, a transplant recipientsuffering from graft versus host disease or graft rejection may betreated by administering Tr1 cells to the recipient to reduce, inhibitor eliminate the graft versus host disease wherein the Tr1 cells areadministered in an amount effective to reduce or eliminate graft versushost disease.

In an embodiment of the invention, the recipient's Tr1 cells may beobtained from the recipient prior to the transplantation and may bestored and/or expanded in culture to provide a reserve of Tr1 cells insufficient amounts for treating an ongoing graft versus host reaction.However, as discussed elsewhere herein, Tr1 cells can be obtained fromany source, for example, from the tissue donor, the transplant recipientor an otherwise unrelated source (a different individual or speciesaltogether).

The skilled artisan will understand that the compositions and methodsdescribed herein can be used in conjunction with current therapeuticapproaches for treating the diseases and disorders described elsewhereherein. By way of non-limiting example, the Tr1 cells of the presentinvention can be used in conjunction with the use of immunosuppressivedrug therapy. An advantage of using Tr1 cells in conjunction withimmunosuppressive drugs is that by using the methods of the presentinvention to ameliorate the severity of the immune response in asubject, such as a transplant recipient, the amount of immunosuppressivedrug therapy used and/or the frequency of administration ofimmunosuppressive drug therapy can be reduced. A benefit of reducing theuse of immunosuppressive drug therapy is the alleviation of generalimmune suppression and unwanted side effects associated withimmunosuppressive drug therapy.

It is also contemplated that the Tr1 cells of the present invention maybe administered into a recipient repeatedly or as a “one-time” therapyfor the prevention or treatment of a disease or disorder, such as anautoimmune disease or disorder, an inflammatory disease or disorder, ora disease or disorder associated with transplant, such as host rejectionof donor tissue or graft versus host disease. A one-time administrationof Tr1 cells into the recipient of the transplant eliminates the needfor chronic immunosuppressive drug therapy. However, if desired,multiple administrations of Tr1 cells may also be employed.

The invention described herein also encompasses a method of preventingor treating transplant rejection and/or graft versus host disease byadministering Tr1 cells in a prophylactic or therapeutically effectiveamount for the prevention, treatment or amelioration of host rejectionof the transplant and/or graft versus host disease. Based upon thepresent disclosure, a therapeutic effective amount of Tr1 cells is anamount that inhibits or decreases the number of activated T cells, whencompared with the number of activated T cells in the absence of theadministration of Tr1 cells. In the situation of host rejection of thetransplant, an effective amount of Tr1 cells is an amount that inhibitsor decreases the number of activated T cells in the recipient of thetransplant when compared with the number of activated T cells in therecipient prior to administration of the Tr1 cells.

An effective amount of Tr1 cells can be determined by comparing thenumber of activated T cells in a subject with a disease or disorderprior to the administration of Tr1 cells thereto, with the number ofactivated T cells present in the subject following the administration ofTr1 cells thereto. A decrease, or the absence of an increase, in thenumber of activated T cells in the subject, or in the transplant itself,that is associated with the administration of Tr1 cells thereto,indicates that the number of Tr1 cells administered is a therapeuticeffective amount of Tr1s.

It should be understood that the methods described herein may be carriedout in a number of ways and with various modifications and permutationsthereof that are well known in the art. It may also be appreciated thatany theories set forth as to modes of action or interactions betweencell types should not be construed as limiting this invention in anymanner, but are presented such that the methods of the invention can bemore fully understood.

These methods described herein are by no means all-inclusive, andfurther methods to suit the specific application will be apparent to theordinary skilled artisan. Moreover, the effective amount of thecompositions can be further approximated through analogy to compoundsknown to exert the desired effect.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the present invention andpractice the claimed methods. The following working examples therefore,specifically point out the preferred embodiments of the presentinvention, and are not to be construed as limiting in any way theremainder of the disclosure.

Example 1: Co-Expression of CD49b and LAG-3 Identifies Human and MurineTr1 Cells

CD4⁺ T regulatory type 1 (Tr1) cells are induced in the periphery andplay a pivotal role in promoting and maintaining tolerance. The absenceof surface markers that uniquely identify Tr1 cells has limited theirstudy and their clinical application. The studies presented hereindemonstrate that by gene expression profiling of human Tr1 cell clones,the surface markers CD49b and LAG-3, which are stably and selectivelyco-expressed on murine and human Tr1 cells, were identified. Asdescribed herein, the specificity of these markers is proven in twomouse models of inflammation and in peripheral blood of healthyvolunteers. The co-expression of CD49b and LAG-3 enables the isolationof highly suppressive human Tr1 cells from in vitro anergized culturesand, enables tracking Tr1 cells in the peripheral blood of tolerantsubjects. As well as being an important finding for the biology of Tr1cells, the identification of these markers makes Tr1 cells an even moreattractive tool for therapeutic interventions.

The materials and methods employed in these experiments are nowdescribed.

Mice

C57BL/6 mice (B6), C57BL/6, RAG1−/− mice, C57BL/6 CD45.1⁺and C57BL/6IL-4^(eGFP) (4get) mice were purchased from The Jackson Laboratories.Dominant Negative IL-10R mice (Pacciani et al., 2010, J Allergy ClinImmunol 125, 727-736), Foxp3 reporter mice (Wan and Flavell, 2005, ProcNatl Acad Sci USA 102, 5126-5131), IL-17A^(eGFP) reporter mice(Esplugues et al., 2011, Nature 475, 514-518), IL-10^(eGFP) reportermice (Kamanaka et al., 2006, Immunity 25, 941-952) and IFN-γ^(Katushka)reporter mice were crossed and generated. Age- and sex-matchedlittermates between 8 and 12 weeks of age were used.

Cell Isolation and Purification of Human Cells

Human peripheral blood from healthy donors (HDs) was obtained uponinformed consent in accordance with local ethical committee approval(TIGET PERIBLOOD) and with the Helsinki Declaration. PBMC were isolatedby centrifugation over Lymphoprep Ficoll gradients (Fresenius Kabi NorgeAS, Halden, Norway). CD4⁺ T lymphocytes were purified from PBMC bynegative selection using the untouched CD4⁺ T Cell Isolation Kit II(Miltenyi Biotech, Auburn, Calif.), according to manufacturer'sinstructions. Naïve CD4⁺CD45RO⁻ T lymphocytes were purified from CD4⁺ Tlymphocytes by CD45RO MicroBeads (Miltenyi Biotech). The proportion ofCD4⁺CD45RO⁻CD45RA⁺ was consistently greater than 90%.

Isolation of Human T Cell Clones

T cell clones were obtained from CD4⁺cells by limiting dilution at 0.3cells/well in the presence of a feeder cell mixture and soluble anti-CD3mAbs (1 μg/mL, OKT3, Jansen-Cilag, Raritan, NJ, USA), in X-vivo 15medium (BioWhittaker, Verviers, Belgium) supplemented with 5% pooledhuman AB serum (BioWhittaker), 100 U/mL penicillin/streptomycin(BioWhittaker). At day 3, IL-2 (40 U/mL; Chiron, Italia, Milan, Italy)was added. T cell clones were re-stimulated every 14 days with feedercell mixture and soluble anti-CD3 mAbs (1 μg/mL). Between stimulationswith feeder cells, T cell clones were expanded with rhIL-2 (40 U/mL).Once the T cell clones had been established, rhIL-15 (5 ng/mL, R&DSystem, Minneapolis, Minn., USA) was added at every change of medium asa Tr1 cell growth factor (Serafini et al., 2009, Haematologica 94,1415-1426; Bacchetta et al., 2002, Eur J Immunol 32, 2237-2245). Theclones were classified based on the cytokine production profile(Romagnani, 1994, Annual review of immunology 12, 227-257). Tr1 cellclones were defined when the ratio between IL-10 and IL-4 was higherthan 8, as previously described (Serafini et al., 2009, Haematologica94, 1415-1426; Bacchetta et al., 2002, Eur J Immunol 32, 2237-2245). AllT cell clones were tested in a suppression assay to assess theirregulatory activity.

T Cell Line Differentiation

Human T Cells

Human Tr1 and T_(H)0 cell lines were differentiated using murine L cellstransfected with hCD32, hCD80, and hCD58 and supplemented with anti-CD3mAb (100 ng/ml; OKT3, Jansen-Cilag, Raritan, NJ, USA) (artificial APCs),as previously described (Levings et al., 2001, J Immunol 166,5530-5539). Briefly, CD4⁺CD45RO″ T cells were activated by previouslyplated irradiated (7000 rad) L cells in X-vivo 15 medium (BioWhittaker)supplemented with 5% pooled human AB serum (BioWhittaker), 100 U/mLpenicillin/streptomycin (BioWhittaker). T_(H)0 cell lines weredifferentiated in the presence of rhIL-2 (100 U/ml; Chiron Italia) andrhIL-15 (1 ng/ml; R&D Systems, Minneapolis, Minn., USA), whereas Tr1cells were polarized with rhIL-10 (100 U/ml; BD Pharmingen), andrhIFNα-2b (5 ng/ml; IntronA, Schering Plough Europe, Bruxelles,Belgium). After 7 days, T cells were re-stimulated under identicalconditions for additional 7 days. At the end of the 14 days of culture,T cells were washed, counted, and analyzed for cytokine production.IL-10-producing T cells were purified by IL-10-secretion assay (MiltenyiBiotech), according to the manufacturer's instruction.

DC-10 was differentiated as previously described (Gregori et al., 2010,Blood 116, 935-944). Briefly, CD14⁺monocytes were isolated as theadherent fraction of PBMC following incubation for 1 hour in RPMI 1640(Biowhittaker) supplemented with 10% FCS (Biowhittaker), 100 U/mlpenicillin/streptomycin (Bristol-Myers Squibb), and 50 μM 2mercaptoethanol (BioRad) (DC medium) at 37° C. Following washing,adherent monocytes were cultured in 10 ng/ml rhIL-4 (R&D Systems) and100 ng/ml rhGM-CSF (R&D Systems) in DC medium in the absence (mDC) orpresence (DC-10) of 10 ng/ml of rhIL-10 (BD, Bioscience) for 7 days.After 5 days, mDC differentiated in the absence of rhIL-10 werestimulated with 1 μg/ml of LPS (Sigma Aldrich) for additional 2 days. Togenerate T(DC-10) cell lines, 10⁵ DC-10 were cultured with 10⁶allogeneic CD4⁺CD45RO⁻ T cells in 1 ml of X-vivo 15 medium(Biowhittaker), supplemented with 5% pooled AB human serum(Biowhittaker), and 100 U/ml penicillin/streptomycin (Bristol-MyersSquibb). After 6 or 7 days, rhIL-2 (20 U/ml; Chiron Italia) was added,and the cells were expanded for additional 7-8 days. Fourteen days afterculture the T cells were collected, washed, and functionally analyzed.As control, T cells differentiated with mDC were used. T cellsstimulated with DC-10 are indicated as pTr1(DC-10), and T cellsstimulated with mDC as T(mDC).

Murine T Cells

Murine naive CD4⁺ T cells (CD4⁺CD62L^(hi)CD25⁻) from C57BL/6 mice wereactivated with plate-bound anti-CD3 (2-5 μg/ml; 145-2C11) and anti-CD28(1-2 μg/ml; PV-1) mAbs. T_(H)0 cells were differentiated in the presenceof anti-IFN-γ (10 μg/ml) and anti-IL-4 (10 μg/ml) mAbs. Tr1 cells weredifferentiated in the presence of murine recombinant IL-27 (25 ng/ml)and TGF-β (2 ng/ml). T_(H)2 cells were differentiated in the presence ofmurine recombinant IL-4 (10 ng/ml) and anti-IFNγ (10 μg/ml). T_(H)17cells were differentiated in the presence of murine recombinant TGF-β(0.5 ng/ml), IL-6 (10 ng/ml), IL-23 (20 ng/ml), anti-IFNγ (10 μg/ml),and anti-IL-4 (10 μg/ml). T_(H)1 cells were differentiated in thepresence of murine recombinant IL-12 (10 ng/ml), IL-2 (50 u/ml), andanti-IL-4 (10 μg/ml). Foxp3⁺Tregs cells were differentiated in thepresence of murine recombinant TGF-β (2 ng/ml), IL-2 (50 U/ml),anti-IFNγ (10 μg/ml) and anti-IL-4 (10 μg/ml). After four days ofculture, T cells were harvested and analyzed.

RNA Isolation and DNA Microarray Experiments

RNA was isolated from Tr1 and T_(H)0 cell clones from two distinct HDsunstimulated (t0) or stimulated (6 and 16 hours) with immobilizedanti-CD3 mAb (10 pg/mL; Jansen-Cilag) and soluble anti-CD28 mAb (1pg/mL, BD Pharmingen) in complete medium at a concentration of 10⁶ Tcells/ml. Total RNA was extracted with RNeasy Mini kit (Qiagen, Hilden,Germany) according to manufacturer's instructions. A total RNA (100 ng)was used for GeneChip analysis. Preparation of terminal-labelled cDNA,hybridization to the whole-transcript GeneChip® Human Gene 1.0 ST Array(Affymetrix, Santa Clara, Calif., USA) and scanning of the arrays wascarried out according to manufacturer's protocols. Raw data waspreprocessed with RMA algorithm. In order to detect differentiallyexpressed genes, Welch t-test without p-value correction was performed.Genes were considered as differentially expressed if gene expression wasmore than 2 times different with p-value <0.05. All these steps wereperformed using R and Bioconductor.

Real-Time Quantitative PCR Analysis

Human Samples

Total RNA was extracted with RNeasy Mini kit (Qiagen, Hilden, Germany),and cDNA was synthesized with high-capacity cDNA Reverse Transcriptionkit (Applied Biosystems, Foster City, Calif.) according tomanufacturer's instructions. Real time analysis was performed using ABIPrism 7500/SDS2.2.1 software. Levels of mRNA were quantified using Assayon Demand quantitative Reverse Transcription Polymerase Chain Reaction(RT-PCR) kits (Applied Biosystems) with TaqMan Universal PCR Master Mix(Applied Biosystems). Samples were run in duplicate or triplicate, andrelative expression was determined by normalizing to hypoxanthinephosphoribosyltransferase 1 (HPRT) and/or β2-microglobulin (B2M)expression in each set of samples to calculate a fold-change in valueand by comparing the relative amount to calibrator (expression level ofa pool of CD4⁺ T cell lines from 4 distinct HDs). Analyses wereperformed with the qBase v1.3.5 software (Jan Hellemans & JoVandesompele).

Murine Samples.

Total RNA was extracted from cells using Trizol® Reagent, followed byRNA clean up using the RNeasy Kit (Quiagen). The High capacity cDNAsynthesis Kit (Applied Biosystems) was used for synthesis of cDNA.Real-time PCR analysis was performed using TaqMan® Fast Universal PCRMater Mix and TaqMan® Gene Expression Assays (Applied Biosystems) on7500 Fast Real-time PCR system machine (Applied Biosystems). Sampleswere run in duplicate or triplicate, and relative expression wasdetermined by normalizing to hypoxanthine phosphoribosyltransferase 1(hrpt) expression.

Cytokine Detection

Human Samples

Human T cells (0.2-0.4×10⁶ cells/ml) were stimulated with immobilizedanti-CD3 mAb (10 μg/mL; Jansen-Cilag) and soluble anti-CD28 mAb (1μg/mL, BD Pharmingen) in complete medium. To measure IL-2, IL-4, IL-10,IFN-γ, and IL-17 production, culture supernatants were harvested after24 (for IL-2 detection), or 72 hours (for other cytokines) of cultureand levels of cytokines were determined by capture ELISA according tothe manufacturer's instruction (BD Biosciences). The limits of detectionwere as follows: IFN-γ: 60 pg/ml; IL-10: 19 pg/ml; IL-4: 9 pg/ml; IL-17:30 pg/ml.

Murine Samples

Murine T cells (0.3-0.5×10⁶ cells/ml) were stimulated for 72 hours withimmobilized anti-CD3 mAb (10 μg/mL; Jansen-Cilag) and soluble anti-CD28mAb (10 μg/mL, BD Pharmingen) in complete medium. Cytokines werequantified by Cytometric Bead Array (BD Bioscience) according to themanufacturer's instructions.

Flow Cytometry Analysis

Human T Cells.

Human T cells were stained with anti-CD4 (BD Pharmingen), anti-CD49b(Biolegend, San Diego, Calif., USA), anti-LAG-3 (R&D System), anti-CD226(Biolegend), anti-CD45RA, and anti-CD25 (BD Pharmingen) mAbs. Thestaining for CD49b and LAG-3 was performed at 37° C. for 15 minutes.Intracellular staining was used for the detection of FOXP3 (clone 259D,Biolegend), following the manufacturers' instructions. Samples wereacquired using a BD FACS Canto flow cytometer (BD Biosciences), and datawas analyzed with FCS express (De Novo Software). Quadrant markers wereset accordingly to unstained controls.

Murine T Cells

Murine T cells were stained with anti-CD4, anti-TCRβ, anti-CD45.1,anti-CD45.2, anti-CD49b (clone HMa2), anti-LAG-3 (clone C9B7W),anti-CD226 mAbs all purchased from Biolegend. The staining for CD49b andLAG-3 was performed at 37° C. for 15 minutes and at room temperature foradditional 15 minutes. For the purification of T cell populationsaccording to the expression of CD49b and LAG-3, CD4⁺ T cells were firstenriched by magnetic-activated cell sorting beads (MACS; MiltenyiBiotec) and then further purified with a FACSVantage (BD). Purity ofsorted cells was higher than 95%.

Suppressive Functions

Human T Cells

To evaluate the suppressive activity of human T cells, CD4⁺ T cells(responder cells) were stained with CFSE (Molecular Probes) and wereactivated with anti-CD3, anti-CD2, and anti-CD28-coated beads (Tr1Inspector, Miltenyi Biotech, Bergisch Gladbach, Germany), at a ratio ofthree beads per cell. Suppressor cells were added at a ratio of 1:1. Thepercentage of divided responder T cells was calculated by gating onCD4⁺cells, as described elsewhere (Lyons and Parish, 1994, J ImmunolMethods 171, 131-137).

Murine T Cells.

To determine the suppressive activity of murine T cells,CD45.1⁺CD4⁺CD25⁻ T cells (responder cells) were labeled with Cell TraceViolet Cell Proliferation Kit (1 μM; Invitrogen) and were cultured in a96-well flat bottom plates (20-50×10³ cells/well) with or withoutCD4⁺CD49b⁺LAG-3⁺Foxp3^(RFP−), CD4⁺CD49b⁻LAG-3⁺Foxp3^(RFP−),CD4⁺CD49b⁺LAG-3⁻Foxp3^(RFP−) and CD4⁺CD49b⁻LAG-3⁻Foxp3^(RFP−) T cellsFACS-sorted from the different organs. The ratio between responder andsuppressor was 1:1, 2:1, 4:1, 8:1. Irradiated APCs (splenocytes MACSdepleted for CD4 and CD8 T cells) were used as feeder cells (4×10⁵cells/well). Cells were stimulated with 1 μg/ml of CD3 mAb (2C11). Insome experiments suppression was performed in the presence ofanti-IL10Rα (50 ug/ml; clone 1B1) mAb. After 72 hours, Cell Trace Violetdilution in CD45.1⁺CD4⁺(responder cells) was analyzed by flow cytometry.The percentage of divided responder T cells was calculated as described(Lyons and Parish, 1994, J Immunol Methods 171, 131-137).

Endoscopic and Histopathology Procedure

Colonoscopy was performed in a blinded fashion for colitis scoring viathe Coloview system (Karl Storz, Germany) (Huber et al., 2011, Immunity34, 554-565; Becker et al., 2006, Nature 440, 303-307). In brief,colitis scoring was based on granularity of mucosal surface, stoolconsistence, vascular pattern, translucency of the colon, and fibrinvisible (0-3 points for each). For the histology, colons were fixed inBouin's fixative solution and embedded in paraffin.

Anti-CD3 and Intestinal Lymphocyte Isolation

Mice were injected with anti-CD3 (15 μg, 145-2C11) mAb, isotypeantibody, or PBS i.p. two times every other day. After removal of thePeyer's Patches, intraepithelial lymphocytes (IEL) and lamina proprialymphocytes (LPL) were isolated via incubation with 5 mM EDTA at 37° C.for 30 min (for IEL), followed by further digestion with collagenase IVand DNase at 37° C. for 1 hour (for LPL). Cells were then furtherseparated with a Percoll gradient. If not indicated differently, cellswere isolated from the upper part of the small intestine(duodenum+jejunum) of anti-CD3-treated mice.

Parasite and Infection

Third-stage larvae (L3) of N. brasiliensis were recovered fromcoprocultures of infected rats and washed extensively. Five hundredparasites were injected subcutaneously in 0.2 ml PBS at the base of thetail, as previously described (Fowell and Locksley, 1999, Bioessays 21,510-518). Mice were sacrificed at designated times, and the presence ofadult worms in the intestines was assessed by inverted microscopy. Wholelungs, spleens, mesenteric and mediastinal lymph nodes were excised,minced, and dispersed into single-cell suspensions. Lung suspensionswere further purified by centrifugation over Ficoll (Brown et al., 1996,J Exp Med 184, 1295-1304).

Patients

Patients affected by β-thalassemia with age ranged from 2 to 17 yearshave been transplanted from HLA-identical sibling donors at the SanRaffaele Scientific Institute since 2005 and at the IstitutoMediterraneo IME since 2004. Eleven patients who developed persistentmixed chimerism (PMC), in which patient and donor cells co-exist forlonger than 2 years after transplantation, and seven patients whodeveloped complete chimerism (CC) after allogeneic HSCT were analyzed.Informed consent was obtained from patients according to institutionalguidelines and to the Helsinki Declaration.

Statistical Analysis

Average values are reported as Mean±SEM. Mann Whitney test and ANOVAtest were used to determine the statistical significance of the data.Significance was defined as *P≤0.05, **P≤0.005, ***P≤0.0005, and***P<0.0001. Statistical calculations were performed with the Prismprogram 5.0 (GraphPad Software, Inc.). Accuracy of the percentages ofCD49b⁺LAG-3⁺ T cells was quantified to discriminate tolerant versusnon-tolerant subjects by Receiver Operating Characteristic (ROC)analysis by means of Area Under Curve (AUC) measurements. To establishthe best screening power of the biomarkers, the “best” cut-off wasinvestigated, which differentiates cases (tolerant subjects) fromcontrols (HDs or non-tolerant subjects). Different cut-offs were chosenand the corresponding sensitivity (proportion of PMC subjects claimed tobe tolerant) and specificity (proportion of HDs or CC subjects claimedto be controls) was computed. Empirical and smoothed ROC curve were thusplotted and compared to the “theoretical” situation with sensitivity andspecificity equal to one. The “best” cut-off was chosen in order tomaximize the “observed” specificity and sensitivity and such thatpercentage of positive cells separates the best cases from controls.Analyses were performed with R 2.15.2 statistical software(R-project.org/R).

The results of the experiments are now described.

Gene Expression Profile of Human Tr1 Cell Clones

The transcriptome of human Tr1 cell clones were compared to that ofT_(H)0 cell clones either unstimulated or stimulated for 6 and 16 h. Thehigh expression of IL-10 (Groux et al., 1997, Nature 389, 737-742), GzB(Magnani et al., 2011 Eur J Immunol 41, 1652-1662; Serafini et al.,2009, Haematologica 94, 1415-1426; Grossman et al., 2004, Blood 104,2840-2848) and PD-1 (Akdis et al., 2004, J Exp Med 199, 1567-1575) (FIG.7A) known to be expressed in Tr1 cells, validated the microarrayaccuracy. The profiles of Tr1 and T_(H)0 cells were similar overall(FIG. 1A), but a small number of transcripts were uniquely expressed inTr1 cell clones (FIG. 1A). Seventeen differentially expressed genes(DEGs) were identified in Tr1 as compared to T_(H)0 cell clones at alltime points, and 28 DEGs upon activation (FIG. 1B, C). Among the 17 DEGsidentified in both unstimulated and stimulated Tr1 cells ITGA2 (CD49b)and CD226 were selected according to the p-values and Log 2FC (FIG. 7B).As CD49b can be expressed also on effector T_(H) cells (Charbonnier etal., 2006, J Immunol 177, 3806-3813; Boisvert et al., 2010, Eur JImmunol 40, 2710-2719), another marker was sought, which, in associationwith CD49b, could allow the isolation of Tr1 cells. LAG-3 (FIG. 7B),which has previously been shown to be associated with Tr1 functions(Workman and Vignali, 2005, J Immunol 174, 688-695) was selected, whichwas highly up-regulated in activated Tr1 cell clones. RT-PCR confirmedthat CD49b, CD226, and LAG-3 were significantly higher in Tr1 thanT_(H)0 cell clones (FIG. 7C), and in enriched IL-10-producing Tr1 celllines isolated from in vitro Tr1-polarized cultures, as compared toT_(H)0 cell lines (FIG. 7D). FACS-analysis confirmed that Tr1 cellclones expressed significantly higher levels of CD49b and LAG-3 thanT_(H)0 cell clones (FIG. 1D). All T cell clones expressed CD226, but Tr1cell clones showed higher mean fluorescence intensity (MFI) than T_(H)0cell clones (FIG. 1D). Overall, CD49b, CD226, and LAG-3 were identifiedas putative markers for human Tr1 cells.

Co-Expression of CD49b and LAG-3 Identifies Human Tr1 Cells

The presence of human CD4⁺ T cells expressing CD49b, LAG-3 and CD226 wasnext investigated. A small population (2.14±0.25%) of memory CD45RA⁻CD4⁺T cells co-expressing CD49b, LAG-3 (FIG. 2A), and CD226 (FIG. 8A) wasobserved in the peripheral blood of healthy donors (HDs). Of note,CD4⁺CD49b⁺LAG-3⁺ T cells did not express CD25 at high levels and theexpression of FOXP3 at mRNA and protein levels was significantly lowerthan in CD25^(bright) T cells (FIG. 8B).

CD4⁺CD49b⁺LAG-3⁺ T cells, FACS-sorted from peripheral blood of HDs,secreted significantly higher levels of IL-10 compared toCD4⁺CD49b⁻LAG-3⁺, CD4⁺CD49b⁺LAG-3⁻, and CD4⁺CD49b⁻LAG-3⁻ T cells, aswell as low amounts of IL-4 (FIG. 2B). CD4⁺CD49b⁺LAG-3⁺ T cellsdisplayed a high IL-10/IL-4 ratio, which is one of the key parameters todistinguish Tr1 from T_(H)2 cells (Groux et al., 1997, Nature 389,737-742; Magnani et al., 2011 Eur J Immunol 41, 1652-1662; Serafini etal., 2009, Haematologica 94, 1415-1426; Passerini et al., 2011, Eur JImmunol 41, 1120-1131), (FIGS. 2B and 8C). Moreover, CD4⁺CD49b⁺LAG-3⁺ Tcells secreted IFN-γ, but not IL-17 (FIG. 2B).

Importantly, CD4⁺CD49b⁺LAG-3⁺ T cells suppressed the proliferation ofCD4⁺ T cells in vitro, which is a key feature of Tr1 cells, atsignificantly higher levels than the other subsets analysed (FIG. 2C).

As demonstrated herein, CD4⁺CD49b⁺LAG-3⁺ T cells represent asubpopulation of CD4⁺memory T cells that secrete high amounts of IL-10,do not express high levels of FOXP3, and exert suppressive activity invitro.

Co-Expression of CD49b and LAG-3 Identifies Murine Tr1 Cells

It was recently shown that CD4⁺Foxp3⁻IL-10⁺ T (Tr1) cells with strongregulatory functions accumulate in the small intestine of mice uponanti-CD3 mAb treatment (Huber et al., 2011, Immunity 34, 554-565). Here,it was tested whether these murine Tr1 cells (defined as CD4⁺TCRβ⁺Foxp3^(RFP−)IL-10^(eGFPbright)) express CD49b and LAG-3. The largemajority (70±5%) of CD4⁺IL-10^(eGFPbright) T cells co-expressed CD49band LAG-3 (FIG. 3A), whereas less than 13±5% of CD4⁺IL-10⁻ T cells wereCD49b⁺LAG-3⁺(FIG. 3A). In line with this finding, CD4⁺CD49b⁺LAG-3⁺ Tcells isolated from the small intestine of anti-CD3 treated micecontained a very high frequency of IL-10^(eGFP+)cells (FIGS. 3B and 3C),and expressed high MFI for IL-10^(eGFP+)(FIG. 3B) and CD226 (FIG. 9A).These results indicate that IL-10-producing T cells and CD4⁺CD49b⁺LAG-3⁺T cells are largely superimposable. Accordingly, the frequencies of CD4⁺TCRβ⁺Foxp3^(RFP−)IL-10^(eGFPbright) and CD4⁺ TCRβ⁺CD49b⁺LAG-3⁺ T cellsshowed the same kinetics after anti-CD3 mAb treatment (FIG. 9B).Notably, the phenotype of Tr1 cells was stable, as CD49b and LAG-3 werepermanently co-expressed by CD4⁺Foxp3⁻IL-10^(eGFPbright) T cells (FIG.9B).

To determine whether CD49b and LAG-3 can be used to isolate murine Tr1cells, CD4⁺CD49b⁺LAG-3⁺ T cells were FACS-sorted and characterized.Without in vitro re-stimulation, CD4⁺CD49b⁺LAG-3⁺ T cells expressed highlevels of Il10 and very low levels of Il4; expression of Ifng, Il2,Tnfa, and Il17a was significantly lower than in T_(H)1 and T_(H)17cells, respectively (FIG. 9C). Upon re-stimulation in vitro,CD4⁺CD49b⁺LAG-3⁺ T cells secreted large amounts of IL-10, which werefive to eight fold higher than IL-4, IL-17A, IL-2, and TNF-α (FIGS. 3Dand 9D), and significant amounts of IFN-γ (FIG. 3D).

CD4⁺CD49b⁺LAG-3⁺ T cells expressed Tbx21, Rorc, and Foxp3 atsignificantly lower levels than T_(H)1, T_(H)17, and Foxp3⁺ Treg cells,respectively. Gata3 levels were similar to those of T_(H)1 and Foxp3⁺Treg cells (FIG. 10). Despite the expression of LAG-3, CD4⁺CD49b⁺LAG-3⁺T cells expressed low levels of Egr2, a transcription factor criticallyinvolved in the development of IL-10-producing LAG-3⁺ Tr1 cells (Okamuraet al., 2009, Proc Natl Acad Sci USA 106, 13974-13979). The expressionof Ahr, a key transcription factor for IL-10 production by Tr1 cells(Apetoh et al., 2010, Nat Immunol 11, 854-861), was significantly higherin CD4⁺CD49b⁺LAG-3⁺ T cells compared to the other cell subsets analyzed(FIG. 10).

CD4⁺Foxp3^(P-)CD49b⁺LAG-3⁺ T cells from the small intestine of anti-CD3treated mice suppressed effector T cells in a dose-dependent manner invitro (FIGS. 4A and 11). Furthermore, using a T cell transfer IBD model(Huber et al., 2011, Immunity 34, 554-565) (FIG. 4B), it wasdemonstrated that CD4⁺Foxp3^(RFP−)CD49b⁺LAG-3⁺ T cells suppressed thecolitogenic eT_(H)17 cells in vivo (FIGS. 4C, 4D, and 4E), in an IL-10dependent manner (FIG. 12).

It was previously shown that Tr1 cells accumulated in the spleen oftolerant pancreatic islet transplanted mice (Battaglia et al., 2006,Diabetes 55, 40-49; Gagliani et al., 2011, PLoS One 6, e28434). In thespleen of anti-CD3 treated mice a population of CD4⁺CD49b⁺LAG-3⁺ T cellswas found that contained a high frequency of IL-10^(eGFP+)cells (FIGS.13A and 13B), displayed a Tr1-cytokine profile (FIG. 13C), andsuppressed T-cell responses in vitro in a partially IL-10-dependentmanner (FIG. 13D).

As demonstrated herein, CD4⁺CD49b⁺LAG-3⁺ T cells, which accumulate inthe intestine and spleen of anti-CD3 treated mice, produce large amountsof IL-10 and have strong suppressive activity in vitro and in vivo. Theco-expression of CD49b and LAG-3 on CD4⁺ T cells, therefore identifiesTr1 cells not only in humans but also in mice.

Co-Expression of CD49b and LAG-3 Distinguishes Tr1 from Other T_(H)Cells.

To test the specificity of CD49b and LAG-3 as markers for Tr1 cells, theexpression of these markers was analysed on other T_(H) cells.

IL-4^(eGFP) reporter mice were infected with N. brasiliensis to examineT_(H)2 cells. In this model the larvae enter the lung 2-3 days aftersubcutaneous injection causing haemorrhage and massive inflammation(Chen et al., 2012, Nat Med 18, 260-266) (FIGS. 14A and 14B). Within9-10 days the adult worms are expelled due to the development ofT_(H)2-type responses Wills-Karp et al., 2012, J Exp Med 209, 607-622;Mohrs et al., 2001, Immunity 15, 303-311). In the present study, it isshown that the majority of T_(H)2 (CD4⁺IL-4^(eGFP+)) cells present indraining lymph nodes (LNs) (FIGS. 5A, and 5D) and in the lungs (FIGS.14C and 14E) did not co-express CD49b and LAG-3.

N. brasiliensis infection also induces a strong IL-17 response in thelungs, which contributes to inflammation and tissue damage (Chen et al.,2012, Nat Med 18, 260-266). It was observed that both T_(H)17(CD4⁺Foxp3^(RFP−)IL-17A^(GFP+)) and Foxp3⁺ Tregs(CD4⁺Foxp3^(RFP+)IL-17A^(GFP−)) cells were induced by N. brasiliensis.These cells accumulated in the draining LNs and in the lungs and did notco-express CD49b and LAG-3 (FIGS. 5A, 5C, 14C, and 14E).

To further prove that T_(H)17 cells do not co-express CD49b and LAG-3,these cells were isolated from the colon of the previously described IBDmodel (Huber et al., 2011, Immunity 34, 554-565). ColitogenicFoxp3^(RFP−)IL-17A^(eGFP+) cells, which include T_(H)17 and asignificant proportion of ‘T_(H)1+T_(H)17’ cells (Huber et al., 2011,Immunity 34, 554-565), and CD4⁺Foxp3^(RFP−)IL-17A^(eGFP−) T cells, whichcontained almost 30-40% of IFN-γ-producing T_(H)1 cells, expressedCD49b, but not LAG-3 (FIGS. 14D and 14E). Furthermore, colitogenicT_(H)1 (Foxp3^(RFP−)IFN-γ^(Katushka+)) cells did not co-express CD49band LAG-3 (FIGS. 14D and 14E).

Thus, as demonstrated herein, unlike Tr1 cells, T_(H)1, T_(H)2, T_(H)17,and Foxp3⁺ Treg cells do not co-express CD49b and LAG-3 in vivo.

During the late phase of N. brasiliensis infection (day 10post-infection) IL-10 production increases and contributes to theresolution of inflammation and consequently tissue damage (Chen et al.,2012, Nat Med 18, 260-266), suggesting the induction of Tr1 cells.CD4⁺Foxp3⁻IL-10⁺ T cells were found in the draining LNs and lung of N.brasiliensis infected mice (FIGS. 5B, 5C, 14C and 14E). The largemajority of CD4⁺Foxp3^(RFP−)IL-10^(GFPbright) T cells wereCD49b⁺LAG-3⁺(FIG. 5B and FIG. 14C). Moreover, CD4⁺ T cells co-expressingCD49b and LAG-3 contained the highest frequency of IL-10^(eGFP+)cellswith the highest MFI (FIGS. 5D, and 5E). CD4⁺CD49b⁺LAG-3⁺ T cellsFACS-sorted from draining LNs of infected mice expressed high levels of1110 mRNA (FIG. 15S) and suppressed the proliferation of effector CD4⁺ Tcells in vitro (FIG. 5F). Notably, during helminth infection in whichthe concentration of T_(H)2-type cytokines is particularly enhanced ininnate and adaptive cells, CD4⁺CD49b⁺LAG-3⁺ T cells expressed Il4, Il13,and Gata3 mRNA at levels comparable to those expressed by Foxp3⁺ Tregcells, but significantly lower than those in T_(H)2 cells (FIG. 15A).Expression of Ahr in CD4⁺CD49b⁺LAG-3⁺ T cells was high but notselective.

Seven days after N. brasiliensis infection Tr1 cells accumulated both inthe lungs and draining LNs (FIGS. 15B and 15C), which is in line withthe described role of IL-10 during resolution of infection (Chen et al.,2012, Nat Med 18, 260-266). The frequency of Tr1 cells (FIGS. 15B and15C) decreased in infected mice over time, but CD49b and LAG-3 werestably co-expressed by CD4+Foxp3^(RFP−)IL-10^(GFPbright) cells (FIGS.15B and 15C).

The expression and stability of CD49b and LAG-3 on Tr1 cells was alsoconfirmed in Tr1 cells differentiated in vitro with IL-27 and TGF-β. Tr1cells expressed Il10 and AhR at significantly higher levels than invitro differentiated T_(H)1, T_(H)2, T_(H)17 and iTreg cells (FIG. 16A).Similar to CD4⁺CD49b⁺LAG-3⁺ T cells from the small intestine of anti-CD3treated mice, expression of Erg2, Gata3, Rorct, Tbx21, and Foxp3 was lowor undetectable in in vitro-induced Tr1 cells (FIG. 16A). Interestingly,the majority of IL-27-induced Tr1 cells were CD49b⁺LAG-3⁺(FIGS. 16B and16C) and the expression of CD49b/LAG-3 remained stable in vitro onIL-10-producing Tr1 cells (FIGS. 17A and 17B). Notably, after in vivotransfer, Tr1 cells that maintained IL-10 expression stably remainedCD49b⁺LAG-3⁺cells (FIGS. 18A and 18B).

Thus, the studies presented herein demonstrate that CD49b and LAG-3 areselectively and stably co-expressed by IL-10-producing Tr1 cells, butnot by T_(H)1, T_(H)2, T_(H)17, and Foxp3⁺ Treg cells.

Clinical Application of Tr1 Cell Specific Surface Markers

To generate Tr1 cells in vitro for therapeutic use, human T cells werepolarized in the presence of IL-10. The resulting cell populationcontains only a small proportion of Tr1 cells and is contaminated by alarge fraction of non-IL-10-producing T cells (Bacchetta et al., 2010,Haematologica 95, 2134-2143). Using previously described protocols todifferentiate human Tr1 cells in vitro (Magnani et al., 2011 Eur JImmunol 41, 1652-1662; Levings et al., 2001, J Immunol 166, 5530-5539;Gregori et al., 2010, Blood 116, 935-944; Gregori et al., 2011, Methodsin molecular biology 677, 31-46), it was shown that the frequency of Tcells co-expressing CD49b and LAG-3 was significantly higher inTr1-polarized cells (FIG. 6A and FIGS. 6B and 19A), compared to T_(H)0cells. FACS-sorted CD49b⁺LAG-3⁺ T cells from Tr1-polarized cellssecreted significantly higher levels of IL-10 (FIG. 6C) and displayedhigher suppressive capacity relative to the original bulk population(FIGS. 6D and 19B), indicating that CD49b and LAG-3 can be used topurify Tr1 cells from in vitro polarized cells.

The frequency of CD49b⁺LAG-3⁺ T cells was assessed in a unique cohort ofβ-thalassemic subjects in which persistent mixed chimerism (PMC) ofdonor and host cells after allogeneic HSCT correlates with tolerance andthe presence of circulating CD4⁻IL-10⁺cells(Serafini et al., 2009,Haematologica 94, 1415-1426). Circulating CD49b⁺LAG-3⁺ T cells weresignificantly higher in peripheral blood of subjects with PMC (Andreaniet al., 2011, Chimerism 2, 21-22; Andreani et al., 2011, Haematologica96, 128-133) compared to both HDs or subject with complete chimerism(CC) (FIGS. 6E and 6F). The statistical analysis confirmed that thepercentage of CD49b⁺LAG-3⁺ T cells can be used to discriminate tolerantsubjects from controls (HDs or CC) (FIGS. 20A and 20B). These findingsdemonstrate that the concomitant expression of CD49b and LAG-3 allowsthe isolation of Tr1 cells from in vitro Tr1-polarized populations andto trace Tr1 cells in vivo in tolerant subjects.

Co-Expression of CD49b and LAG-3b

The studies presented herein demonstrate that co-expression of CD49b andLAG-3 identifies human and murine Tr1 cells. CD4⁺CD49b⁺LAG-3⁺ T cellssecrete large amounts of IL-10, display a high IL-10/IL-4 andIL-10/IL-17 ratio, express high levels of CD226, do not express highFoxp3 and possess strong IL-10-dependent regulatory activity.Concomitant expression of CD49b and LAG-3 is specific for Tr1 cells,since T_(H)1, T_(H)2, T_(H)17 and Foxp3⁺ Treg cells do not co-expressthese markers. Co-expression of CD49b and LAG-3 can be used to purifyhuman Tr1 cells from in vitro Tr1-polarized cell cultures, and enablestracing of Tr1 cells in tolerant subjects.

Expression of CD49b has been previously described on effector memoryCD4⁺ T cells (Kassiotis et al., 2006, J Immunol 177, 968-975), T_(H)17cells (Boisvert et al., 2010, Eur J Immunol 40, 2710-2719) andIL-10-producing T cells (Charbonnier et al., 2006, J Immunol 177,3806-3813; Rahmoun et al., 2006, Int Arch Allergy Immunol 140, 139-149).The present data shows that CD49b is expressed on Tr1 cells, but also onT_(H)1, T_(H)2, T_(H)17 cells and Foxp3⁺ Treg cells. LAG-3 is expressedon splenic T cells isolated from naïve mice with regulatory function andcorrelates with IL-10 production (Okamura et al., 2009, Proc Natl AcadSci USA 106, 13974-13979; Huang et al., 2004, Immunity 21, 503-513).However, activated T cells also express LAG-3 (Workman and Vignali,2005, J Immunol 174, 688-695; Bettini et al., 2011, J Immunol 187,3493-3498; Bruniquel et al., 1998, Immunogenetics 48, 116-124; Lee etal., 2012, Nat Immunol 13, 991-999; Huard et al., 1997, Proc Natl AcadSci USA 94, 5744-5749). It is shown that murine and human T cellsexpressing LAG-3 but not CD49b produce IL-4, low amounts of IL-10, arehighly proliferative, and do not display significant suppressiveactivity in vitro.

Thus, the use of either CD49b alone or LAG-3 alone, is not sufficient toselect a highly enriched population of functional Tr1 cells, or todistinguish these cells from other Tx or Treg cell subsets. It isdemonstrated herein that the combination of CD49b and LAG-3 is requiredto identify and select murine and human Tr1 cells, which secrete highlevels of IL-10 and have regulatory activity in vitro and in vivo. BothCD49b and LAG-3 are stably expressed on functional Tr1 cells. CD49b isexpressed by Tr1 cells irrespectively of their activation, whereas LAG-3is expressed on Tr1 cells when they produce IL-10 and display suppressoractivity. Co-expression of CD49b and LAG-3 distinguishes Tr1 cells fromT_(H)1, T_(H)2, T_(H)17 cells during helminth infection and IBD.

The identification of Tr1 cells in patients has been limited by theirability to produce IL-10 only upon in vitro re-stimulation (Bacchetta etal., 1994, J Exp Med 179, 493-502; Meiler et al., 2008, J Exp Med 205,2887-2898; Petrich de Marquesini et al., 2010, Diabetologia 53,1451-1460; Sanda et al., 2008, Clin Immunol 127, 138-143). Moreover,intracellular flow cytometric analysis of IL-10 expression isinsensitive and is highly variable according to the type of stimuli.Alternatively, T-cell cloning of circulating CD4⁺ T cells allows theenumeration of IL-10-producing Tr1 cells in tolerant subjects (Bacchettaet al., 1994, J Exp Med 179, 493-502; Gregori et al., 2011, Methods inmolecular biology 677, 31-46). Using these techniques, it was previouslydemonstrated that high frequencies of IL-10-producing T cells and of Tr1cell clones in peripheral blood of allogeneic HSCT transplanted subjectscorrelated with persistent mixed chimerism and tolerance (Bacchetta etal., 1994, J

Exp Med 179, 493-502; Serafini et al., 2009, Haematologica 94,1415-1426). It is shown herein that in these tolerant subjects thefrequency of CD4⁺CD49b⁺LAG-3⁺ T cells is significantly increased.Statistical analysis shows significant differences in the percentages ofCD49b⁺LAG-3⁺ T cells in tolerant subjects versus control groups. SinceCD49b⁺LAG-3⁺ T cells are IL-10-producing suppressor T cells, these dataindicate that the frequency of Tr1 cells can be monitored in vivo usingthese markers.

Regulatory T cell-based therapies have become an attractive therapeuticoption for inducing/restoring tolerance. Several protocols to generateand expand Tr1 cells in vitro have been developed (Bacchetta et al.,2010, Haematologica 95, 2134-2143; Brun et al., 2009, IntImmunopharmacol 9, 609-613), and proof-of-principle clinical trialsdemonstrating safety and feasibility of Tr1 cell-infusion have beenrecently completed (Bacchetta et al., 2009, Blood, 45 (ASH AnnualMeeting Abstract; Desreumaux et al., 2012, Gastroenterology 143,1207-1217 e1201-1202). However, the cell preparation consisting ofantigen-specific IL-10-anergized T cells generated with recombinantIL-10 or DC-10 (Gregori et al., 2010, Blood 116, 935-944; Bacchetta andGregori, 2010, Hematologica 95, 2134-2143) still contains a subset ofcontaminating non-Tr1 cells, which could potentially exacerbate thepathogenic clinical condition of patients. The data presented hereinshow that CD49b and LAG-3 co-expression allows the isolation of Tr1cells from in vitro Tr1-polarized populations and from antigen-specificIL-10-anergized T cells, thereby rendering their clinical use safer andbroadening their clinical application.

In summary, two selective markers for Tr1 cells that are conserved inmice and humans have been discovered. These markers make it possible tostudy the in vivo localization of Tr1 cells in physiological conditions,as well as the role of Tr1 cells in subjects with immune-mediateddiseases in which a defect in Tr1 cells has been proposed.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

1. A method of treating or preventing a disease or disorder associatedwith an immune response in a subject in need thereof, the methodcomprising administering to the subject an effective amount ofCD4+CD49+LAG-3+ Tr1 cells.
 2. The method of claim 1, wherein the cellsare CD45RA−.
 3. The method of claim 1, wherein the disease or disorderis an inflammatory disease or disorder.
 4. The method of claim 1,wherein the disease or disorder is an autoimmune disease or disorder. 5.The method of claim 1, wherein the disease or disorder is associatedwith transplantation.
 6. The method of claim 1, wherein the disease ordisorder is at least one selected from the group consisting of allergy,asthma, inflammatory bowel disease, autoimmune entheropathy, Addision'sdisease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis,autoimmune parotitis, Crohn's disease, diabetes mellitus, dystrophicepidermolysis bullosa, epididymitis, glomerulonephritis, Graves'disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia,systemic lupus erythematosus, multiple sclerosis, myasthenia gravis,pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis,sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies,thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia,ulcerative colitis, cell and organ transplant rejection and graft versushost disease.
 7. The method of claim 1 for treating an inflammatorydisease or disorder, an autoimmune disease or disorder, and a disease ordisorder associated with transplantation in a subject in need thereof.8. A method of inhibiting alloreactive T cells in a subject in needthereof, the method comprising contacting the alloreactive T cells withan effective amount of CD4+CD49+LAG-3+ Tr1 cells.
 9. The method of claim8, wherein the cells are CD45RA−.
 10. A method for preventing ortreating an immune response in a subject in need thereof, said methodcomprising administering to said subject prior to onset of the immuneresponse an effective amount of CD4⁺CD49⁺LAG-3⁺ Tr1 cells to preventsaid response.
 11. The method of claim 10, wherein the cells areCD45RA−.
 12. The method of claim 10, wherein the immune response is analloreactive response.
 13. The method of claim 10, wherein the immuneresponse is an inflammatory response.
 14. The method of claim 10,wherein the immune response is an autoimmune response.